PD1 and/or LAG3 BINDERS
United States Patent Application
The present invention provides molecules, such as ISVDs and Nanobodies, that bind to PD1 and LAG3 and, optionally to human serum albumin. These molecules have been engineered so as to reduce the incidence of binding by pre-existing antibodies in the bodies of a subject administered such a molecule. Methods for increasing immune response, treating cancer and/or treating an infectious disease with such molecules are provided.
Inventors:
Bowman, Edward (Redwood City, CA, US)
Beaumont, Maribel (San Mateo, CA, US)
Buyse, Marie-ange (Merelbeke, BE)
Boutton, Carlo (Wielsbeke, BE)
Dombrecht, Bruno (Heusden, BE)
Vlerick, David (Gentbrugge, BE)
Application Number:
Publication Date:
05/18/2017
Filing Date:
11/17/2016
Export Citation:
Bowman Edward
Beaumont Maribel
Buyse Marie-Ange
Boutton Carlo
Dombrecht Bruno
Vlerick David
Primary Class:
International Classes:
C07K16/28; A61K39/395; A61K45/06; C07K16/46
View Patent Images:
&&&&&&PDF help
Related US Applications:
June, 2008Li et al.December, 2007DeathMay, 2009Chen et al.August, 2009Hotz et al.December, 2006LacyDecember, 2004ChambersJune, 2005Soane et al.September, 2005ValenzuelaMay, 2007Pak et al.September, 2008Nierop Groot et al.October, 2005Cremer et al.
Attorney, Agent or Firm:
MERCK (P O BOX 2000
RAHWAY NJ )
A PD1 binder comprising an immunoglobulin single variable domain (ISVD) that binds to PD1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2 but comprising one or more mutations at a residue selected from 11 and 89; wherein said residue numbers are K and, optionally, a half-life extender and/or a C-terminal extender.
A PD1 binder comprising one or more immunoglobulin single variable domains (ISVDs) that binds to PD1 comprising: a CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); a CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V); and a CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F); comprising one or more mutations at a residue selected from 11 and 89, wherein said residue numbers are Kabat residue numbers.
The PD1 binder of claim 1 wherein the immunoglobulin single variable domain comprises the amino acid sequence: DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57, optionally comprising a DILE mutation); EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO: 98, optionally comprising a E1D mutation); EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO: 99, optionally comprising a E1D mutation); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 103, optionally comprising a D1E and/or N73S mutation); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 104, optionally comprising a D1E mutation); or DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 105, optionally comprising a D1E mutation), or DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (amino acids 1-19 of SEQ ID NO: 113, optionally comprising a D1E mutation); or DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (amino acids 1-19 of SEQ ID NO: 117, optionally comprising a D1E mutation); or DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (amino acids 1-19 of SEQ ID NO: 121, optionally comprising a D1E mutation); and, optionally, comprising a half-life extender and/or a C-terminal extender.
A LAG3 binder comprising an immunoglobulin single variable domain (ISVD) that binds to LAG3 comprising a CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65) or DYVMG (amino acids 6-10 of SEQ ID NO: 65); a CDR2 comprising the amino acid sequence AISESGGRTHYADSVKG (SEQ ID NO: 66) or AISESGGRTH (amino acids 1-10 of SEQ ID NO: 66); and a CDR3 comprising the amino acid sequence TLLWWSEYAPIKANDYDY (SEQ ID NO: 67); and, optionally, a half-life extender and/or a C-terminal extender.
The LAG3 binder of claim 4 wherein the immunoglobulin single variable domain comprises the amino acid sequence: EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64); optionally comprising a E1D mutation, and optionally, comprising a half-life extender and/or a C-terminal extender.
A multispecific binder comprising an immunoglobulin single variable domain (ISVD) that binds to PD1 comprising: a CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); a CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V); and a CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F); and an immunoglobulin single variable domain that binds to LAG3 comprising: a CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65) or DYVMG (amino acids 6-10 of SEQ ID NO: 65); a CDR2 comprising the amino acid sequence AISESGGRTHYADSVKG (SEQ ID NO: 66) or AISESGGRTH (amino acids 1-10 of SEQ ID NO: 66); and a CDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67); and, optionally, a half-life extender and/or a C-terminal extender.
The multispecific binder of claim 6 wherein the immunoglobulin single variable domain that binds to PD1 comprises the amino acid sequence: DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57); EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO: 98); EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO: 99); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (amino acids 1-119 of SEQ ID NO: 101); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDNSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 103); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 104); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNT VYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 105); the immunoglobulin single variable domain that binds to LAG3 comprises the amino acid sequence: EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64); or DVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREFVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLR PEDTALYYCATTLLWWTSEYAPIKANDYDYWGQGTLVTVSS (amino acids 1-128 of SEQ ID NO: 96); and, optionally, a half-life extender and/or a C-terminal extender.
The binder of claim 1 comprising a C-terminal extender wherein the C-terminal extender is an alanine.
The multispecific binder of claim 6 comprising: a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 98; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 98; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 63; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 63; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 99; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 20 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 99; a 20 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 20 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 20 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 9 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 99; a 9 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9 GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 20GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 20GS linker comprising the amino acid sequence set forth in SEQ ID NO: 100; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 57; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105 (D1E); a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105 (D1E); a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 105; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104 (D1E); a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104 (D1E); a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 103 (N73S); a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:103 (D1E, N73 S); a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 103 (N73S); a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:103 (D1E, N73 S); a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 103 (N73S); a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine or a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 103 (N73S); a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forth in SEQ ID NO: 59; and a C-terminal Alanine.
The multispecific binder of claim 6 comprising the amino acid sequence selected from SEQ ID NOs: 106-124.
The multispecific binder of claim 1 which is bound to PD1 and/or LAG3.
The multispecific binder of claim 1 comprising a half-life extender wherein the half-life extender is a human serum albumin ISVD that binds to human serum albumin comprising: CDR1 comprising the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60) or SFGMS (amino acids 1-5 of SEQ ID NO: 60); CDR2 comprising the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL (amino acids 1-10 of SEQ ID NO: 61); and CDR3 comprising the amino acid sequence GGSLSR (SEQ ID NO: 62).
The multispecific binder of claim 12 wherein the human serum albumin ISVD comprises the amino acid sequence: (SEQ ID NO: 59)EVQLVESGGG VVQPGNSLRL SCAASGFTFS SFGMSWVRQAPGKGLEWVSS ISGSGSDTLYADSVKGRFTI SRDNAKTTLY LQMNSLRPED TALYYCTIGG SLSRSSQGTL VTVSSA
A binder that cross-blocks a binder of claim 1 from binding to LAG3 or PD1 and LAG3.
An injection device or vessel that comprises the binder of claim 1, optionally in association with a further therapeutic agent.
A polynucleotide encoding the binder of claim 1.
A vector comprising the polynucleotide of claim 16.
A host cell comprising the polynucleotide of claim 16.
A method for making a binder of claim 1 comprising introducing a polynucleotide encoding the binder into a host cell and culturing the host cell in a medium under conditions favorable to expression of said binder from said polynucleotide and, optionally, purifying the binder from said host cell and/or said medium.
A binder produced by the method of claim 19.
A method for preventing PD1 from binding to PD-L1 and/or PD-L2 comprising contacting the PD1 with said binder of claim 1 optionally in association with a further therapeutic agent.
A method for preventing LAG3 from binding to MHC class II comprising contacting said LAG3 with a binder of claim 4, optionally in association with a further therapeutic agent.
A method for enhancing an immune response in a subject comprising administering an effective amount of binder of claim 1 to the subject, optionally in association with a further therapeutic agent.
A method for treating or preventing cancer or an infectious disease in a human subject comprising administering an effective amount of binder of claim 1, optionally in association with a further therapeutic agent, to the subject.
The method claim 24 wherein the cancer is metastatic cancer, a solid tumor, a hematologic cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer.
The method claim 24 wherein the infectious disease is a bacterial infection, a viral infection or a fungal infection.
The method of claim 24 wherein the subject is administered a further therapeutic agent or a therapeutic procedure in association with the binder.
Description:
This Application claim the benefit of U.S. Provisional Patent Application No. 62/257,009, filed Nov. 18, 2015; which is herein incorporated by referenced in its entiretyA computer readable format nucleotide/amino acid Sequence Listing is incorporated by reference in its entirety. The file containing the Sequence Listing is a 199 kbyte ASCII text file created Nov. 15, 2016 named “24238WOPCTSEQ”.FIELD OF THE INVENTIONThe present invention relates, in part, to amino acid sequences binding to programmed cell death protein 1 (“PD1”), e.g., human PD1 and lymphocyte activation gene 3 (LAG3). In particular, the present invention relates, in part, to improved heavy-chain immunoglobulin single variable domains (also referred to herein as “ISVs” or “ISVDs”) binding to PD1 and LAG3, as well as to polypeptides and other compounds that comprise such ISVDs. Other aspects, embodiments, features, uses and advantages of the invention will be clear to the skilled person based on the disclosure herein.BACKGROUND OF THE INVENTIONProgrammed death receptor 1 (PD-1) is an immunoinhibitory receptor that is primarily expressed on activated T and B cells. Interaction with its ligands has been shown to attenuate T-cell responses both in vitro and in vivo. Blockade of the interaction between PD-1 and one of its ligands, PD-L1, has been shown to enhance tumor-specific CD8+ T-cell immunity and may therefore be helpful in clearance of tumor cells by the immune system.PD-1 (encoded by the gene Pdcd1) is an Immunoglobulin superfamily member related to CD28, and CTLA-4. PD-1 has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2) The structure of murine PD-1 has been solved as well as the co-crystal structure of mouse PD-1 with human PD-L1 (Zhang, X. et al., Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 08)). PD-1 and like family members are type I transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail that is responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, an ITIM (immunoreceptor tyrosine-based inhibition motif) and an ITSM (immunoreceptor tyrosine-based switch motif).Following T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3 zeta, PKC theta and ZAP70 that are involved in the CD3 T cell signaling cascade. The mechanism by which PD-1 downmodulates T cell responses is similar to, but distinct from that of CTLA-4, as both molecules regulate an overlapping set of signaling proteins (Parry et al., Mol. Cell. Biol. 25:
(2005)). Bennett and coworkers have shown that PD-1-mediated inhibition of T-cell signaling is only effective when both activating and inhibitory signals are on the same surface, indicating that the PD-1 signaling mechanism is spatiotemporally determined (Bennett F. et al., J Immunol. 170:711-8 (2003)). PD-1 was shown to be expressed on activated lymphocytes (peripheral CD4+ and CD8+ T cells, B cells and monocytes) and has also been shown to be expressed during thymic development on CD4-CD8- (double negative) T cells as well as NK-T cells.The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as various tumor types. PD-L1 is expressed on B, T, myeloid and dendritic cells (DCs), but also on peripheral cells, like microvascular endothelial cells and non-lymphoid organs like heart, lung etc. In contrast, PD-L2 is only found on macrophages and DCs. The expression pattern of PD-1 ligands is suggestive of a role for PD-1 in maintaining peripheral tolerance and may serve to regulate self-reactive T- and B-cell responses in the periphery. Both ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region. Both ligands contain short cytoplasmic regions with no known signaling motifs.Interaction of PD-1 with its ligands leads to the inhibition of lymphocyte proliferation in vitro and in vivo. Disruption of the PD-1/PD-L1 interaction has been shown to increase T cell proliferation and cytokine production and block progression of the cell cycle. Initial analysis of Pdcd1-/- mice did not identify any drastic immunological phenotype. However aged mice developed spontaneous autoimmune diseases which differ according to the strain onto which the Pdcdl deficiency was backcrossed. These include lupus-like proliferative arthritis (C57BL/6) (Nishimura H. et al., Int. Immunol. 10:
(1998)), fatal cardiomyopathy (BALB/c) (Nishimura H. et al., Science 291: 319-322 (2001)) and type I diabetes (NOD) (Wang J. et al., Proc. Natl. Acad. Sci. USA 102:
(2005)). Overall, analysis of the knockout animals has led to the understanding that PD-1 functions mainly in inducing and regulating peripheral tolerance. Thus, therapeutic blockade of the PD-1 pathway may be helpful in overcoming immune tolerance. Such selective blockade may be of use in the treatment of cancer or infection as well as in boosting immunity during vaccination (either prophylactic or therapeutic).The role of PD-1 in cancer is established in the literature. It is known that tumor microenvironment can protect tumor cells from efficient immune destruction. PD-L1 has recently been shown to be expressed on a number of mouse and human tumors (and is inducible by IFN gamma on the majority of PD-L1 negative tumor cell lines) and is postulated to mediate immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99:
(2002); Strome S. E. et al., Cancer Res., 63:
(2003).In humans, expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 (on tumor cells) has been found in a number of primary tumor biopsies assessed by immunohistochemistry. Such tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, oral squamous cell, urothelial cell, and pancreas as well as tumors of the head and neck (Brown J. A. et al., J. Immunol. 170:
(2003); Dong H. et al., Nat. Med. 8: 793-800 (2002); Wintterle et al., Cancer Res. 63:
(2003); Strome S. E. et al., Cancer Res., 63:
(2003); Thompson R. H. et al., Cancer Res. 66: 06); Thompson et al., Clin. Cancer Res. 13: 07); Nomi T. et al., Clin. Cancer Res. 13: 2151-7. (2007)). More strikingly, PD-ligand expression on tumor cells has been correlated to poor prognosis of cancer patients across multiple tumor types (reviewed in Okazaki and Honjo, Int. Immunol. 19: 813-824 (2007)).Blockade of the PD-1/PD-L1 interaction could lead to enhanced tumor-specific T-cell immunity and therefore be helpful in clearance of tumor cells by the immune system. To address this issue, a number of studies were performed. In a murine model of aggressive pancreatic cancer, T. Nomi et al. (Clin. Cancer Res. 13:
(2007)) demonstrated the therapeutic efficacy of PD-1/PD-L1 blockade. Administration of either PD-1 or PD-L1 directed antibody significantly inhibited tumor growth. Antibody blockade effectively promoted tumor reactive CD8+ T cell infiltration into the tumor resulting in the up-regulation of anti-tumor effectors including IFN gamma, granzyme B and perforin. Additionally, the authors showed that PD-1 blockade can be effectively combined with chemotherapy to yield a synergistic effect. In another study, using a model of squamous cell carcinoma in mice, antibody blockade of PD-1 or PD-L1 significantly inhibited tumor growth (Tsushima F. et al., Oral Oncol. 42: 268-274 (2006)).In other studies, transfection of a murine mastocytoma line with PD-L1 led to decreased lysis of the tumor cells when co-cultured with a tumor-specific CTL clone. Lysis was restored when anti-PD-L1 mAb was added (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99:
(2002)). In vivo, blocking the PD1/PD-L1 interaction was shown to increase the efficacy of adoptive T cell transfer therapy in a mouse tumor model (Strome S. E. et al., Cancer Res. 63:
(2003)). Further evidence for the role of PD-1 in cancer treatment comes from experiments performed with PD-1 knockout mice. PD-L1 expressing myeloma cells grew only in wild-type animals (resulting in tumor growth and associated animal death), but not in PD-1 deficient mice (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99:
(2002)).In human studies, R. M. Wong et al. (Int. Immunol. 19:
(2007)) showed that PD-1 blockade using a fully human anti-PD-1 antibody augmented the absolute numbers of tumor-specific CD8+ T cells (CTLs) in ex vivo stimulation assays using vaccine antigens and cells from vaccinated individuals. In a similar study, antibody blockade of PD-L1 resulted in enhanced cytolytic activity of tumor-associated antigen-specific cytotoxic T cells and increased cytokine production by tumor specific TH cells (Blank C. et al., Int. J. Cancer 119: 317-327 (2006)). The same authors showed that PD-L1 blockade augments tumor-specific T cell responses in vitro when used in combination with anti-CTLA-4 blockade.Overall, the PD-1/PD-L1 pathway is a well-validated target for the development of antibody therapeutics for cancer treatment. Anti-PD-1 antibodies may also be useful in chronic viral infection. Memory CD8+ T cells generated after an acute viral infection are highly functional and constitute an important component of protective immunity. In contrast, chronic infections are often characterized by varying degrees of functional impairment (exhaustion) of virus-specific T-cell responses, and this defect is a principal reason for the inability of the host to eliminate the persisting pathogen. Although functional effector T cells are initially generated during the early stages of infection, they gradually lose function during the course of a chronic infection. Barber et al. (Barber et al., Nature 439: 682-687 (2006)) showed that mice infected with a laboratory strain of LCMV developed chronic infection resulting in high levels of virus in the blood and other tissues. These mice initially developed a robust T cell response, but eventually succumbed to the infection upon T cell exhaustion. The authors found that the decline in number and function of the effector T cells in chronically infected mice could be reversed by injecting an antibody that blocked the interaction between PD-1 and PD-L1.Recently, it has been shown that PD-1 is highly expressed on T cells from HIV infected individuals and that receptor expression correlates with impaired T cell function and disease progression (Day et al., Nature 443:350-4 (2006); Trautmann L. et al., Nat. Med. 12: 06)). In both studies, blockade of the ligand PD-L1 significantly increased the expansion of HIV-specific, IFN-gamma producing cells in vitro.Other studies also implicate the importance of the PD-1 pathway in controlling viral infection. PD-1 knockout mice exhibit better control of adenovirus infection than wild-type mice (Iwai et al., J. Exp. Med. 198:39-50 (2003)). Also, adoptive transfer of HBV-specific T cells into HBV transgenic animals initiated hepatitis (Isogawa M. et al., Immunity 23:53-63 (2005)). The disease state of these animals oscillates as a consequence of antigen recognition in the liver and PD-1 upregulation by liver cells.In addition, LAG3 (CD223) is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39:213-217, 1994), NK cells (Triebel et al. J Exp Med 171:, 1990), B cells (Kisielow et al. Eur J Immunol 35:, 2005), and plasmacytoid dendritic cells (Workman et al. J Immunol 182:, 2009) that plays an important role in the function of these lymphocyte subsets. In addition, the interaction between LAG3 and its major ligand, Class II MHC, is thought to play a role in modulating dendritic cell function (Andreae et al. J Immunol 168:, 2002). Recent preclinical studies have documented a role for LAG-3 in CD8 T-cell exhaustion (Blackburn et al. Nat Immunol 10:29-37, 2009).As with chronic viral infection, tumor antigen-specific CD4+ and CD8+ T cells display impaired effector function and an exhausted phenotype characterized by decreased production of pro-inflammatory cytokines and hyporesponsiveness to antigenic re-stimulation. This is mediated by cell extrinsic mechanisms, such as regulatory T-cells (Treg), and cell intrinsic mechanisms, such as inhibitory molecules that are upregulated on exhausted, tumor-infiltrating lymphocytes (TIL). These inhibitory mechanisms represent a formidable barrier to effective antitumor immunity.LAG-is expressed on tolerized TILs suggesting that they contribute to tumor-mediated immune suppression. Inhibition of LAG3 may lead to enhanced activation of antigen-specific T cells from which a therapeutic benefit may be gained.SUMMARY OF THE INVENTIONThe present invention encompasses a PD1 binder (e.g., an immunoglobulin single variable domain (ISVD) or a Nanobody that binds to PD1 (e.g., human PD1) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2; but which comprises a mutation at one or more of positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and 112 wherein said positions are numbered according to Kabat (e.g., L11V, A14P, A74S, K83R, I89L and, optionally, E1D; or L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100a and, optionally, E1D), comprising CDR1 that comprises the amino acid sequence: IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2 that comprises the amino acid sequence: VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V); and CDR3 that comprises the amino acid sequence: DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F) or DKHQSSXYDY (SEQ ID NO: 8, wherein X is W or F). In an embodiment of the invention, the PD1 binder (e.g., an ISVD such as a Nanobody) comprises an amino acid residue at position 11 that is chosen from L or V; an amino acid residue at position 89 chosen from T, V or L; an amino acid residue at position 110 chosen from T, K or Q; and/or an amino acid residue at position 112 chosen from S, K or Q. In an embodiment of the invention, the PD1 binder (e.g., an ISVD such as a Nanobody) comprises one or more mutations described is a member selected from the group consisting of: 89T; 89L in combination with 11V; 89L in combination with 110K or 110Q; 89L in combination with 112K or 112Q; 89L in combination with 11V and 110K or 110Q; 89L in combination with 11V and 112K or 112Q; 11V in combination with 110K or 110Q; 11V in combination with 112K or 112Q. In an embodiment of the invention, the amino acid at positions 11, 89, 110 and 112 are as any of those set forth in the Table B herein. In an embodiment of the invention, the PD1 binder (e.g., an ISVD such as a Nanobody) comprises one or more mutations at a position selected from the group consisting of 1, 14, 41, 74, 83, 87 and 108 and/or one or more humanizing substitutions known per se (for which reference is made to the prior art cited herein, such as WO 08/020079 or WO 09/138519. In an embodiment of the invention, the PD1 binder (e.g., an ISVD such as a Nanobody) comprises a C-terminal extension of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. For example, in an embodiment of the invention, the PD1 binder (e.g., an ISVD such as a Nanobody) comprises a C-terminal extension according to the formula —X(n), wherein X and n are as follows: (a) n=1 and X=A (b) n=2 and each X=A (c) n=3 and each X=A (d) n=2 and at least one X=Ala and wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(e) n=3 and at least one X=Ala and wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(f) n=3 and at least two X=Ala and wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(g) n=1 and X=G (h) n=2 and each X=G (i) n=3 and each X=G (j) n=2 and at least one X=Gly wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(k) n=3 and at least one X=Gly wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(1) n=3 and at least two X=Gly wherein the remaining amino acid residue(s) X are independently chosen from any naturally
(m) n=2 and each X=Ala or G (n) n=3 and each X=Ala or G (o) n=3 and at least one X=Ala or Gly wherein remaining amino acid residue(s) X are independently chosen from any naturally
or (p) n=3 and at least two X=Ala or Gly wherein the remaining amino acid residue(s) X are independently chosen from any naturally occurring amino acid, e.g., A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA or GAG. The present invention also provides a PD1 binder (e.g., an ISVD such as a Nanobody) comprising one or mutations at position 11, 89, 110 and 112 and an amino acid sequence having at least 85% (e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9 or 100%) sequence identity with the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 9-40 (in which any C-terminal extension that may be present as well as any CDRs are not taken into account for determining the degree of sequence identity). The present invention also provides a multispecific immunoglobulin comprising a PD1 binder (e.g., an ISVD such as a Nanobody) that binds to PD1 linked to one or more molecules that bind to an epitope that is not the epitope to which the PD1 binder binds (e.g., PD1, CTLA4, LAG3, BTLA and/or CD27), e.g., comprising a PD1 binding moiety and a CTLA4 a PD1 binding moiety and a BTLA a PD1 binding moiety and a LAG3 or a PD1 binding moiety, a LAG3 binding moiety and a BTLA binding moiety, optionally linked via one or more linkers, e.g., peptide linkers. In one embodiment of the invention, the multispecific immunoglobulin comprises a first PD1 Nanobody linked to one or more molecules selected from the group consisting of an CTLA4 Nanobody, an LAG3 Nanobody, an BTLA Nanobody, an CD27 Nanobody and an PD1 Nanobody that binds to the same or a different epitope as that of the first PD1 N e.g., comprising a PD1 binding moiety and a CTLA4 a PD1 binding moiety and a BTLA a PD1 binding moiety and a LAG3 or a PD1 binding moiety, a LAG3 binding moiety and a BTLA binding moiety.The present invention includes a polypeptide, ISVD or Nanobody comprising an amino acid sequence that is described herein, e.g., which is selected from SEQ ID NOs: 9-40.The present invention also provides a PD1 binder of the present invention (e.g., an immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) in association with a further therapeutic agent.Injection devices and vessels comprising PD1 binder (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) optionally in association with a further therapeutic agent are provided by the present invention.The present invention provides a polynucleotide encoding a PD1 binder (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody); or a vector comprising the polynucleotide or a host cell comprising the polynucleotide or vector.The present invention provides a method for making an PD1 binder (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) comprising introducing a polynucleotide encoding the immunoglobulins into a host cell and culturing the host cell in a medium under conditions favorable to expression of said immunoglobulin from said polynucleotide and, optionally, purifying the immunoglobulin from said host cell and/or said medium. PD1 binders (e.g., an ISVDs such as a Nanobodies) produced by such methods are part of the present invention.The present invention also provides a method for preventing PD1 from binding to PD-L1 and/or PD-L2 comprising contacting said PD1 with a PD1 binder (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) optionally in association with a further therapeutic agent.The present invention also provides a method for enhancing an immune response in the body of a subject comprising administering an effective amount of a PD1 binder of the present invention (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) to the subject optionally in association with a further therapeutic agent. In addition, the present invention also provides a method for treating or preventing cancer (e.g., metastatic cancer, a solid tumor, a hematologic cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer) or an infectious disease (bacterial infection, a viral infection or a fungal infection) in the body of a subject comprising administering an effective amount of PD1 binder (e.g., immunoglobulin single variable domain (ISVD) or multispecific ISVD such as a Nanobody) optionally in association with a further therapeutic agent to the subject. In an embodiment of the invention, the subject is also administered a further therapeutic agent or a therapeutic procedure in association with the PD1 binder.The present invent invention provides PD1 binders (e.g., ISVDs such as Nanobodies) that bind to PD1 comprising: CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V); and CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F), which comprise one or more mutations at position 11 (e.g., L11V) and 89 (e.g., I89L) or one or more mutations selected from E1D, L11V, A14P, W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aF with respect to SEQ ID NO: 1 or 2. In an embodiment of the invention, the mutations are E1D, L11V, A14P, K83R and I89L; or L11V, A14P, K83R and I89L. In an embodiment of the invention, the mutations are E1D, L11V, A14P, W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aF or L11V,A14P,W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aF. In an embodiment of the invention, the PD1 binder comprises the amino acid sequence: DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57).The present invention also provides LAG3 binders that bind to LAG3 comprising CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65); CDR2 comprising the amino acid sequence AISESGGRTH (SEQ ID NO: 66); and CDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67), e.g., comprising the amino acid sequence: EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ H31\10:64).The PD1 and LAG3 binders may be in a single molecule such as a PD1/LAG3 binder, which is part of the present invention, that binds to PD1 and LAG3 that comprises: a PD1 binder comprising: CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V); and CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F); and a LAG3 binder comprising: CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65); CDR2 comprising the amino acid sequence AISESGGRTH (SEQ ID NO: 66); and CDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67); and, optionally, a half-life extender, e.g., wherein the PD1 binder comprises the amino acid sequence: DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTINTVSS; and (SEQ ID NO: 57); and the LAG3 binder comprises the amino acid sequence: EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64); and, optionally, a half-life extender. For example, in an embodiment of the invention, the PD1/LAG3 binder comprises the moieties, e.g., in the order shown:
the PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L) or 1PD102C12 (E1D, L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or N73S), A74S, K83R, I89L, W100aF);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));the LAG3 binder 11B09 or 11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));a half-life extender such as ALB11002; and, optionally,a C-terminal extensio orthe PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L) or 1PD102C12 (E1D, L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or N73S), A74S, K83R, I89L, W100aF);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));the PD1 binder 102C12 (L11V,A14P,A74S,K83R,I89L) or 1PD102C12 (L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or N73S), A74S, K83R, I89L, W100aF);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));the LAG3 binder 11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));the LAG3 binder 11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);a peptide linker such as 9GS, 20GS or 35 GS (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));a half-life extender such as ALB11002; and, optionally,a C-terminal extension such as an alanine.
The half -life extender is, in an embodiment of the invention, a human serum albumin (HSA) binder such as ALB11002. In an embodiment of the invention, the HSA binder comprises CDR1 comprising the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60); CDR2 comprising the amino acid sequence SISGSGSDTL (SEQ ID NO: 61); and CDR3 comprising the amino acid sequence GGSLSR (SEQ ID NO: 62), e.g., comprising the amino acid sequence EVQLVESGGG VVQPGNSLRL SCAASGFTFSS FGMSWVRQA PGKGLEWVSS ISGSGSDTLYADSVKGRFTI SRDNAKTTLY LQMNSLRPED TALYYCTIGG SLSRSSQGTL VTVSSA (SEQ ID NO: 59). Such half-life extenders themselves are part of the present invention.In an embodiment of the invention, the PD1 binder, LAG3 binder, HSA binder and/or PD1/LAG3 binder is in an injection device or vessel optionally in association with a further therapeutic agent. Such a device or vessel is part of the present invention.The present invention also provides a polynucleotide encoding any of the binders set forth herein as well as any vector (e.g., plasmid) comprising the polynucleotide as well as any host cell (e.g., CHO or fungal cell such as Pichia, e.g., P. pastoris) comprising the polynucleotide or vector, e.g., ectopic or integrated into one or more host cell chromosomes.The present invention also provides a method for making any of the binders set forth herein comprising introducing a polynucleotide encoding the binder into a host cell (e.g., CHO or fungal cell such as Pichia, e.g., P. pastoris) and culturing the host cell in a medium under conditions favorable to expression of said binder from said polynucleotide and, optionally, purifying the binder from said host cell and/or said medium. Optionally, the vector or polynucleotide is integrated into one or more host cell chromosomes. Any binder produced by such a method is also part of the present invention.The present invention also provides a method for preventing PD1 from binding to PD-L1 and/or PD-L2 or for inhibiting any PD1 activity, e.g., as discussed herein, comprising contacting said PD1 with a PD1 binder of the present invention (e.g., a PD1/LAG3 binder) optionally in association with a further therapeutic agent. The present invention also provides a method for preventing LAG3 from binding to MHC class II or for inhibiting any PD1 activity, e.g., as discussed herein comprising contacting said LAG3 with a LAG3 binder of the present invention (e.g., a PD1/LAG3 binder) optionally in association with a further therapeutic agent. The present invention also provides a method for enhancing an immune response in the body of a subject (e.g., a human) comprising administering an effective amount of binder of the present invention (e.g., PD1/LAG3 binder) to the subject optionally in association with a further therapeutic agent. The present invention also provides a method for treating or preventing cancer (e.g., metastatic cancer, a solid tumor or a hematologic cancer) or an infectious disease (e.g., bacterial infection, a viral infection or a fungal infection) in the body of a subject (e.g., a human) comprising administering an effective amount of binder of the present invention (e.g., a PD1/LAG3 binder) optionally in association with a further therapeutic agent to the subject. When administered to a subject, a PD1 and/or LAG3 binder of the present invention (e.g., a PD1/LAG3 binder) is optionally administered in association with a further therapeutic agent or a therapeutic procedure e.g., surgical tumorectomy.DESCRIPTION OF THE FIGURESFIG. 1. A table listing some of the amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (such as Aho and IMGT)FIG. 2(1-5). PD1 binder sequences.FIG. 3(A-B). Alignment of 102C12 sequence with that of the SEQ ID NOs: 9-40 (see WO , SEQ ID NO: 348).FIG. 4. Predominant N-linked glycans for monoclonal antibodies produced in Chinese hamster ovary cells (CHO N-linked glycans) and in engineered yeast cells (engineered yeast N-linked glycans): squares: N-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).FIG. 5. Monovalent binding of CHO-K1 expressing human PD1 by F (1PD102C12(A14P,A74S,K83R)-FLAG3-HIS6) or F (1PD102C12 (L11V,A14P,A74S,K83R,I89L)-FLAG3-HIS6). GAM=goat anti-mouse secondary antibody use to detect mouse antibody that binds to FLAG α-FLAG+GAM=goat anti-mouse secondary antibody used plus mouse antibody that binds to FLAG epitope that is at C-terminus of the construct.FIG. 6. Monovalent binding of 3A9 cells expressing human LAG3 by F-FLAG-His6 or F (F(L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-FLAG3-HIS6.FIG. 7(A-B). Binding of (A) CHO expressing human PD-1 by F, F, F or F or (B) 3A9 expressing rhesus PD-1 by F (Pichia or CHO expressed), F (Pichia or CHO expressed), F, F, F (1PD102C12 (A14P,A745,K83R)-35G5-ALB11002) or F. ABH0074+GAM=goat anti-mouse secondary antibody used plus mouse antibody ABH0074 that binds to nanobody frameworks.FIG. 8(A-B). Binding of (A) CHO expressing human LAG3 by F, F or F or (B) 3A9 expressing rhesus LAG3 by F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F.FIG. 9(A-H). Blockage of binding between (A) human PD-L1-Fe and CHO-K1 expressing human PD1 by F or F; (B) human PD-L2-Fc and CHO-K1 expressing human PD1 by F or F; (C) human PD-L1-Fc and CHO-K1 expressing human PD1 by F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F; (D) human PD-L2-Fc and CHO-K1 expressing human PD1 by F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F; (E) human PD-L1-Fc and CHO-K1 expressing human PD1 by F, F, F or F; (F) human PD-L2-Fc and CHO-K1 expressing human PD1 by F, F, F or F; (G) human PD-L1-Fe and CHO-K1 expressing human PD1 by F (1PD102C12 (L11V,A14P,A74S,K83R,I89L)-HIS6), F (1PD102C12 (E1D,L11V,A14P,W52aV,A74S,K83R,I89L,W100aF)-HIS6), F (1PD102C12 (E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6, or F (1PD102C12(E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6); (H) human PD-L2-Fc and CHO-K1 expressing human PD1 by F (1PD102C12 (L11V,A14P,A74S,K83R,I89L)-HIS6), F (1PD102C12 (E1D,L11V,A14P,W52aV,A74S,K83R,I89L,W100aF)-HIS6), F (1PD102C12 (E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6, or F (1PD102C12 (E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6). US=S hPD-L1 EC30=the staining intensity of hPD-L1-Fe that has been titrated to give 30% of the maximal staining intensity th GAH=goat anti-human secondary antibody used to detect the human Fc portion of hPD-L1-Fe.FIG. 10(A-C). Blockade of human LAG3-Fc binding to Daudi cells by (A) F-FLAG3-H156, F-FLAG3-HIS6 or human LAG3-Fc; (B) F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F, human LAG3-Fc; or (C) F, F or F or human LAG3-Fc. Sec only=secondary antibody only (GAH/GANI).FIG. 11(A-B). Proximity Assay (beta-galactosidase enzyme fragment complementation assay system) with (A) F; F, F, control Nanobody (IRR00085; respiratory syncitia virus (RSV) binder), F or F; or (B) F; F, F, control Nanobody (IRR00085), F or F.FIG. 12(A-E). Activation of Jurkat T-cells (expression of luciferase operably linked to IL2 promoter) in presence of HSA by (A) F or control Nanobody (IRR00043; two anti-lysozyme nanobodies linked with a 35GS linker that has a C-terminal FLAG3-His6), (B) F (Pichia or CHO expressed), F (Pichia or CHO expressed), F, F or control Nanobody (IRR00085 or IRR00087; RSV binder), (C) F F, F or control Nanobody (IRR00043), (D) F, F, F or control Nanobody (IRR00043), or (E) F, F (1PD102C12(E1D,L11V,A14P,W52aV,N73Q, A74S,K83R,I89L,W100aF)-HIS6, F (1PD102C12 (E1D,L11V,A14P,W52aV, N73P,A74S,K83R,I89L,W100aF)-HIS6 or control nanobody (IRR00088; RSV binder).FIG. 13(A-R). Activation of human peripheral blood monocytes (IL2 production) from donors (A) 91,(B) 985, (C) 907, (D) 91, (E) 985, (F) 907, (G) 91 (with 10 nM SEB), (H) 985 (with 10 nM SEB), (I) 907 (with 10 nM SEB), (J) 91(with 25 nM SEB), (K) 985 (with 25 nM SEB), (L) 907 (with 25 nM SEB) with F (Pichia or CHO expressed), F (Pichia or CHO expressed), F, F or control N or (M) 91 (N) 985 (O) 907 with F, F or control N or (P) 91, (Q) 985 or (R) 907 with F, F, F.FIG. 14(A-F). Mixed lymphocyte assay of CD4 T-cells and dendritic cells from different donors determining (A-C) interferon-gamma production at varying concentrations of Nanobody F (Pichia or CHO expressed), F (Pichia or CHO expressed) or F o (D-F) interferon-gamma production at varying concentrations of Nanobody F, F, F, F F or F or control Nanobody.FIG. 15(A-D). Activation assay of 3A9 T-cells expressing human LAG3 in the presence of HSA and in the presence of (A) F (11B09 (E1D)), F or control IgG4; (B) F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F or control Nanobody (IRR00085 or IRR00087; RSV binders); (C) F, F, F or control N (D) F, F, or F or control Nanobody.FIG. 16(A-B). Activation of Jurkat T-cells expressing human LAG3 and human PD1 and Raji antigen-presenting cells in the presence of (A) F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F [should this be F] or control Nanobody or (B) F, F, F, F, F, F, F or F or control Nanobody.FIG. 17(A-C). Human T-cell clone activation (interferon-gamma production) in the presence of JY cells expressing human PDL1 in the presence of varying concentrations of (A) F (Pichia or CHO expressed), F (Pichia or CHO expressed), F or F or control N (B) F, F, F, F or F or control N or (C) F, F, F, F or F or control Nanobody.FIG. 18(A-P). Sequences of the present invention.FIG. 19(A-I). Serum preAb reactivity to F and F and a trivalent control Nanobody T (lacking mutations to reduce pre-existing antibody binding) by healthy human subject sera at (A) 125 seconds and (B) 360 by cancer patient sera at (C) 125 seconds or (D) 360 or by sera of patients suffering from (E) melanoma, (F) non-small cell lung cancer (NSCLC), (G) head & neck cancer, (H) gastric cancer or (I) colorectal cancer.DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides ISVDs that comprise mutations which block reactivity of pre-existing antibodies (pre-antibodies) to neo-epitopes within the ISVDs. Neoepitopes are epitopes within a protein which are revealed when the protein is mutated (e.g., truncated) or its folding is altered. Pre-existing antibodies are antibodies existing in the body of a patient prior to receipt of an ISVD. The ISVDs of the present invention are based, in part, in llama antibodies whose C-terminal constant domai thus, exposing the neo-epitopes in the C-terminus of the resulting VHH to pre-antibody binding. It has been discovered that the combination of mutations of residues 11 and 89 (e.g., L11V and I89L or V89L) led to a surprising lack of pre-antibody binding. Mutations in residue 112 have also been shown to remarkably reduce pre-antibody binding. Buyse & Boutton (W) included data showing that the combination of an L 11V and V89L mutation provided a remarkable improvement in reducing pre-antibody binding compared to an L11V mutation alone or a V89L mutation alone. For example, Table H of Buyse & Boutton on page 97 showed comparative data for an ISVD with a V89L mutation alone (with or without C-terminal extension) and the same ISVD with a V89L mutation in combination with an L11V mutation (again, with or without a C-terminal extension). Also, although generated in two separate experiments, the data shown in Table H for the L11V/V89L combination as compared to the data given in Table B for an L11V mutation alone (in the same ISVD) showed that the pre-antibody binding reduction that is obtained by the L11V/V89L combination was greater than that for the L11V mutation alone.Since the llama antibody scaffold structure is known to be very highly conserved, the effect of the mutations at positions 11 and 89 is very likely to exist for any ISVD. Indeed, the effect was demonstrated, in FIG. 19, with the instant binders, F and F, which were shown to exhibit very low levels of pre-antibody binding in healthy subjects and subjects suffering from cancer.In the present application, the amino acid residues/positions in an immunoglobulin heavy-chain variable domain will be indicated with the numbering according to Kabat. For the sake of convenience, FIG. 1 gives a table listing some of the amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (such as Aho and IMGT). This point is also further discussed herein.With regards to the CDRs, as is well-known in the art, there are multiple conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop regions). Reference is for example made to the website www.bioinf.org.uk/abs/. For the purposes of the present specification and claims, even though the CDRs according to Kabat may also be mentioned, the CDRs are most preferably defined on the basis of the Abm definition (which is based on Oxford Molecular's AbM antibody modelling software), as this is considered to be an optimal compromise between the Kabat and Chotia definitions. Reference is again made to the website www.bioinf.org.uk/abs/). See Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883; Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Elvin A. Kabat, Tai Te Wu, Carl Foeller, Harold M. Perry, Kay S. Gottesman (1991) Sequences of Proteins of Immunological I Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In an embodiment of the invention, CDR determination is according to Kabat, e.g., wherein FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113.In an embodiment of the invention, CDRs are determined according to Kontermann and Dithel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010).The term “immunoglobulin single variable domain” (also referred to as “ISV” or ISVD”) is generally used to refer to immunoglobulin variable domains (which may be heavy chain or light chain domains, including VH, VHH or VL domains) that can form a functional antigen binding site without interaction with another variable domain (e.g. without a VH/VL interaction as is required between the VH and VL domains of conventional 4-chain monoclonal antibody). Examples of ISVDs will be clear to the skilled person and for example include Nanobodies (including a VHH, a humanized VHH and/or a camelized VHs such as camelized human VHs), IgNAR, domains, (single domain) antibodies (such as dAbs(TM)) that are VH domains or that are derived from a VH domain and (single domain) antibodies (such as dAbs(TM)) that are VL domains or that are derived from a VL domain. ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, an ISVD will be a Nanobody.The term “Nanobody” is generally as defined in WO 08/020079 or WO 09/138519, and thus in a specific aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g. optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression). It is noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx N.V. and thus may also be referred to as Nanobody(R) and/or Nanobodies(R)). An example of an ISVD is 102C12 (E1D, L11V, A14P, A74S, K83R, I89L). Other ISVDs also appear in Tables A and C herein.A multispecific binder (e.g., multispecific ISVD) is a molecule that comprises, for example, a first PD1 or LAG3 binding moiety (e.g., an ISVD or a Nanobody) and one or more (e.g., 1, 2, 3, 4, 5) additional binding moieties (e.g., an ISVD or a Nanobody) that bind to an epitope other than that of the first PD1 or LAG3 binding moiety (e.g., to CTLA4, CD27 and/or BTLA); e.g., comprising a PD1 or LAG3 binding moiety and a CTLA4 a PD1 or LAG3 binding moiety and a BTLA one or two PD1 binding moieties and one or two LAG3 binding moieties and a human serum al or a PD1 binding moiety, a LAG3 binding moiety and a BTLA binding moiety. A multispecific binder is, for example, F or F.A binding moiety or binding domain or binding unit is a molecule such as an ISVD or Nanobody that binds to an antigen. A binding moiety or binding domain or binding unit may be part of a larger molecule such as a multivalent or multispecific immunoglobulin that includes more than one moiety, domain or unit and/or that comprises another functional element, such as, for example,