EA041987B1 - PD1 AND/OR LAG3 BINDING MOLECULES, METHODS OF THEIR APPLICATION, POLYNUCLEOTIDE, VECTOR AND HOST CELL - Google Patents

Description

This application claims priority under US Provisional Application No. 62/257009, filed Nov. 18, 2015, which is incorporated herein by reference in its entirety.

The list of nucleotide/amino acid sequences in machine readable format is incorporated herein by reference in its entirety. The file containing the sequence list is a 199 KB ASCII text file created on November 15, 2016 named 24238WOPCTSEQ.

Field of invention

The present invention relates in particular to amino acid sequences that bind to programmed cell death protein 1 (PD1), eg human PD1, and to lymphocyte activation gene 3 (LAG3). In particular, the present invention relates, in part, to improved immunoglobulin heavy chain single variable domains (also referred to herein as JSV or ISVD) that bind to PD1 and LAG3, as well as to polypeptides and other compounds that contain such ISVDs. Other aspects, embodiments, features, uses, and advantages of the invention will be apparent to the skilled artisan from the description of the invention provided herein.

Prerequisites for the creation of the invention

Programmed death receptor 1 (PD-1) is an immunoinhibitory receptor that is primarily expressed on activated T and B cells. Its interaction with its ligands has been shown to attenuate the T cell response both in vitro and in vivo. Inhibition 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 therefore may be useful in the elimination of tumor cells PD-1 (encoded by the Pdcd1 gene) is a member of the immunoglobulin superfamily , related to CD28 and CTLA-4. PD-1 has been shown to negatively regulate antigen-receptor signaling when interacting with its ligands (PD-L1 and/or PD-L2). The structure of mouse PD-1 as well as co-crystal structure of mouse PD-1 with human PD-L1 has been solved (Zhang, X. et al., Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci USA 105: 3011-6 (2008)). PD-1 and similar members of the family are type I transmembrane glycoproteins containing an immunoglobulin variable type (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine signaling motifs, ITIM (immunoreceptor tyrosine inhibitory motif) and ITSM (immunoreceptor tyrosine switching motif).

Upon stimulation of T cells, tyrosine phosphatase SHP-2 binds to the ITSM motif within the cytoplasmic tail of PD-1, resulting in dephosphorylation of effector molecules such as CD3-zeta, PKC-theta, and ZAP70, which are involved in the CD3 signaling cascade of T cells. The mechanism by which PD-1 reduces the T cell response is similar, but not completely, to that of CTLA-4, as both molecules regulate an overlapping set of signaling proteins (Parry et al., Mol. Cell. Biol. 25: 9543-9553 (2005)). Bennett et al showed that PD-1 mediated T cell signal inhibition is only effective when activation and inhibition signals are on the same surface, indicating that the PD-1 signaling mechanism is spatiotemporally regulated (Bennett F. et al., J. Immunol., 170: 711-8 (2003)). PD-1 has been 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 maturation in the thymus on CD4- ^CD8 (double negative) T cells, as well as on NK-T cells.

Ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in various cell types, including non-hematopoietic tissues, as well as in various types of tumors. PD-L1 is expressed on B, T, myeloid and dendritic cells (DC), but also on peripheral cells such as microvascular endothelial cells and non-lymphoid organs such as heart, lung, etc. In contrast, PD-L2 is found only on macrophages and DCs. The PD1 ligand expression pattern indicates a role for PD-1 in maintaining peripheral tolerance and may serve to regulate auto 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 signal motifs.

Interaction of PD-1 with its ligands results in inhibition of lymphocyte proliferation in vitro and in vivo. It has been shown that disruption of the PD-1/PD-L1 interaction increases T cell proliferation and cytokine production and blocks the development of the cell cycle. Primary analysis of PdcdT/' mice did not reveal a radical immunological phenotype. However, spontaneous autoimmune diseases occurred in older mice, which differed depending on the strain of mice with which the mice deficient in Pdcd1 were backcrossed. These include lupus-like proliferative arthritis (C57BL/6) (Nishimura H. et al., Int. Immunol., 10: 1563-1572 (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: 11823-11828 (2005)). Overall, analysis of knockout animals led to the understanding that PD-1 functions primarily in the induction and regulation of peripheral

- 1 041987 ransom. Thus, therapeutic blockade of the PD-1 pathway may be useful in overcoming immune tolerance. Such selective blockade may be useful in the treatment of cancer or infection, as well as to increase immunity through vaccination (prophylactic or therapeutic).

The role of PD-1 in cancer has been described in the literature.

It is known that the microenvironment of tumors can protect tumor cells from effective destruction by the immune system. Recently, PD-L1 has been shown to be expressed on a number of mouse and human tumors (and IFN-gamma is induced in most PD-L1-negative tumor cell lines) and postulated to mediate immune surveillance evasion (Iwai Y. et al., USA, 99: 12293-12297 (2002), Strome S. E. et al., Cancer Res., 63: 6501-6505 (2003)).

In humans, expression of PD-1 (on tumor-infiltrating lymphocytes) and/or PD-L1 (on tumor cells) has been detected in a number of primary tumor biopsies, as determined by immunohistochemistry. Such tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, mouth, urothelial cells, and pancreas, as well as tumors of the head and neck (Brown J. A. et al., J. Immunol. 170: 1257-1266 (2003); Dong H. et al., Nat. Med. 8: 793-800 (2002); Wintterle et al., Cancer Res. 63: 74627467 ( 2003); Strome S. E. et al., Cancer Res., 63: 6501-6505 (2003); Thompson R. H. et al., Cancer Res. 66: 3381-5 (2006); Thompson et al., Clin. Cancer Res. 13: 1757-61 (2007) Nomi T. et al., Clin Cancer Res. 13: 2151-7.

(2007)). More importantly, PD ligand expression on tumor cells correlates with poor prognosis in cancer patients having various types of tumors (reviewed in Okazaki and Honjo, Int. Immunol, 19: 813-824 (2007)).

Blockade of the PD-1/PD-L1 interaction may lead to an increase in the tumor-specific T-cell immune response and therefore may be useful in clearing tumor cells by the immune system. To solve this problem, a number of studies have been carried out. In a mouse model of aggressive pancreatic cancer, T. Nomi et al. (Clin. Cancer Res., 13: 2151-2157 (2007)) demonstrated the therapeutic efficacy of PD-1/PD-L1 blockade. Administration of an antibody to either PD-1 or PD-L1 significantly inhibited tumor growth. Inhibition with antibodies effectively increased the infiltration of tumor-specific CD8+ T cells into the tumor, which led to an increase in the synthesis of antitumor effectors, including IFN-gamma, granzyme B, and perforin. In addition, the authors showed that PD-1 blockade can be effectively combined with chemotherapy, giving a synergistic effect. In another study using a mouse model of squamous cell carcinoma, blockade with anti-PD-1 or PD-L1 antibodies significantly inhibited tumor growth (Tsushima F. et al., Oral Oncol., 42: 268-274 (2006)).

In other studies, transfection of a mouse mastocytoma line with PD-L1 resulted in reduced tumor cell lysis when co-cultured with a tumor-specific CTL clone. Lysis was repaired by adding mAb to PD-L1 (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297 (2002)). Blocking the PD1/PD-L1 interaction in vivo has been shown to increase the efficacy of adoptive T cell transfer therapy in a mouse tumor model (Strom S. E. et al., Cancer Res., 63: 6501-6505 (2003)). Further evidence for the role of PD-1 in cancer treatment comes from experiments done with PD-1 knockout mice. Myeloma cells expressing PD-L1 grew only in wild-type animals (leading to 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: 12293-12297 (2002)).

In human studies, PM Wong et al. (Int. Immunol., 19: 1223-1234 (2007)) showed that blockade of PD-1 using a fully human anti-PD-l antibody increased the absolute number of tumor-specific CD8+ T cells ( CTL) in ex vivo stimulation assays using vaccine antigens and cells from vaccinated individuals. In a similar study, blocking PD-L1 with antibodies resulted in increased cytolytic activity of tumor-associated antigen-specific cytotoxic T cells and increased cytokine production by tumor-specific T _H cells (Blank C. et al., Int. J. Cancer 119: 317-327 (2006 )). The same authors showed that PD-L1 blockade enhances tumor-specific T cell responses in vitro when used in combination with CTLA-4 blockade.

Overall, the PD-1/PD-L1 pathway is a well-established target for the development of therapeutic agents for cancer. Anti-PD-1 antibodies may also be useful in chronic viral infection. Memory CD8+ T cells formed 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 (depletion) of virus-specific T cell responses, and this defect is the main reason for the host's inability to eliminate the persistent pathogen. Although functional effector T cells are primarily generated during the early stages of infection, they gradually lose their functionality during chronic infection. Barber et al. (Barber et al., Nature 439: 682-687 (2006)) showed that mice infected with a laboratory strain of LCMV developed a chronic infection resulting in high levels of virus in the blood and other tissues. These mice initially developed strong T

- 2 041987 cellular response, but eventually they developed an infection when T cells were depleted. The authors found that the decrease in the number and function of effector T cells in chronically infected mice can be reversed by injecting antibodies that block the interaction between PD-1 and PD-L1.

It has recently been shown that PD-1 is highly expressed on T cells in 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: 1198-202 (2006)). In both studies, blockade of the PD-L1 ligand significantly increased the expansion of HIV-specific IFN-gamma producing cells in vitro.

Other studies have also shown the importance of the PD-1 pathway in fighting viral infection. PD-1 knockout muscles were better able to control adenovirus infection than wild-type mice (Iwai et al., J. Exp., Med., 198: 39-50 (2003)). In addition, adoptive transfer of HBV-specific T cells into HBV transgenic animals has caused hepatitis (Isogawa M. et al., Immunity 23: 53-63 (2005)). The course of the disease in these animals oscillates as a result of antigen recognition in the liver and an increase in the level of PD-1 in the 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: 1393-1405 , 1990), B cells (Kisielow et al. Eur J Immunol 35: 2081-2088, 2005) and plasmacytoid dendritic cells (W or kman et al. J Immunol 182: 188 5-1891, 2009), which play an important role in the functioning of these subpopulations of lymphocytes. In addition, the interaction between LAG3 and its main ligand, MHC class II, is believed to play a role in modulating dendritic cell function (Andreae et al. J Immunol 168: 3874-3880, 2002). Recent preclinical studies have confirmed the role of LAG-3 in CD8 T cell depletion (Blackburn et al. Nat Immunol 10: 2937, 2009).

As in chronic viral infection, CD4+ and CD8+ tumor antigen-specific T cells exhibit impaired effector function and a depleted phenotype characterized by reduced production of pro-inflammatory cytokines and reduced response to antigen re-stimulation. This process is mediated by extrinsic mechanisms acting on the cell, such as regulatory T cells (Treg), and intracellular mechanisms, such as inhibitory molecules, whose synthesis is increased on depleted, tumor-infiltrating lymphocytes (TILs). These inhibitory mechanisms are a serious obstacle to an effective antitumor immune response.

LAG is expressed on tolerant TILs, indicating that they enhance tumor-mediated immunosuppression. Inhibition of LAG3 can lead to increased activation of antigen-specific T cells, which can lead to a therapeutic effect.

The essence of the invention

The present invention relates to a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD) or nanobody that binds to PD1 (e.g., human PD1)) containing the amino acid sequence shown in SEQ ID NO: 1 or 2, but which contains mutations in one or more positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and 112, where the indicated positions are numbered according to the Kabat system (for example, L11V, A14P, A74S, K83R, I89L and, optionally, E1D, or L11V, A14P, W52aV, N73P, A74S, K83R, I89L, W100a and optionally E1D) containing a CDR1 that contains the amino acid sequence: IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6) , CDR2 which contains the amino acid sequence: VITXSGGITYYADSVKG (SEQ ID NO: 4; where X is W or V) or VITXSGGITY (SEQ ID NO: 7; where X is W or V), and CDR3 which contains the amino acid sequence: DKHQSSXYDY (SEQ ID NO: 5, where X is F and or W) or DKHQSSXYDY (SEQ ID NO: 8, where X is W or V). In an embodiment, a PD1 binding molecule (e.g., an ISVD such as a nanobody) contains an amino acid residue at position 11 selected from L or V, an amino acid residue at position 89 selected from T, V, or L, an amino acid residue at position 110, selected from T, K or Q, and/or amino acid residue at position 112 selected from S, K or Q. In an embodiment, the PD1 binding molecule (e.g. selected from the group consisting of: 89T; 89L in combination with 11V; 89L combined with 110K or 110Q; 89L combined with 112K or 112Q; 8 9L combined with 11V and 110K or 110Q; 89L in combination with 11V and 112K or 112Q; 11V combined with 110K or 110Q; 11V combined with 112K or 112Q. In an embodiment of the invention, the amino acids at positions 11, 89, 110 and 112 are any of those listed in table. in this document. In one embodiment, the PD1 binding molecule (e.g., ISVD, such as a nanobody) contains 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 several humanizing substitutions previously known (for which reference is made to the prototype specified in this document, such as WO 08/020079 or WO 09/138519). In an embodiment, the PD1 binding

- 3 041987 molecule (for example, ISVD, such as nanobodies) includes a C-terminal extension of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. For example, in an embodiment, a PD1 binding molecule (e.g., an ISVD, such as a nanobody) contains a C-terminal extension according to the formula -X(n), where X and n are: (a) n=1 and X=Ala ; (b) n=2 and each X=Ala; (c) n=3 and each X=A1a; (d) n=2 and at least one X=A1a, and where the remaining amino acid residue(s) X is independently selected from any natural amino acid;

(e) n=3 and at least one X=Ala, and where the remaining amino acid residue(s) X is independently selected from any natural amino acid; (f) n=3 and at least two X=Ala, and where the remaining amino acid residue(s) X is independently selected from any natural amino acid;

(g) n=1 and X=Gly; (h) n=2 and each X=Gly; (i) n=3 and each X=Gly;

(j) n=2 and at least one X=Gly, where the remaining amino acid residue(s) X is independently selected from any natural amino acid; (k) n=3 and at least one X=Gly, where the remaining amino acid residue(s) X is independently selected from any natural amino acid; (l) n=3 and at least two X=Gly, where the remaining amino acid residue(s) X is independently selected from any natural amino acid; (m) n=2 and each X=A1a or Gly; (n) n=3 and each X=A1a or Gly; (o) n=3 and at least one X=Ala or Gly, where the remaining amino acid residue(s) X is independently selected from any natural amino acid; or (p) n=3 and at least two X=Ala or Gly, where the remaining amino acid residue(s) X is independently selected from any natural amino acid, e.g. A, AA, AAA, G, GG, GGG, AG, GA , AAG, AGG, AGA, GGA, GAA or GAG. The present invention also relates to a PD1 binding molecule (e.g., an ISVD, such as a nanobody) containing one or more mutations at positions 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 shown in the representative selected from the group 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 in determining the degree of sequence identity). The present invention also provides a multispecific immunoglobulin comprising a PD1-binding fragment (e.g., an ISVD, such as a nanobody) that binds to PD1, coupled to one or more molecules that bind an epitope other than the epitope to which the PD1-binding fragment binds (e.g., , PD1, CTLA4, LAG3, BTLA and/or CD27), for example, containing a PD1-binding fragment and a CTLA4-binding fragment; a PD1 binding fragment and a BTLA binding fragment; a PD1 binding fragment and a LAG3 binding fragment; or a PD1-binding fragment, a LAG3-binding fragment, and a BTLA-binding fragment, optionally connected via one or more linkers, such as peptide linkers. In one embodiment, the multispecific immunoglobulin comprises a first PD1 nanobody associated with one or more molecules selected from the group consisting of a CTLA4 nanobody, a LAG3 nanobody, a BTLA nanobody, a CD27 nanobody, and a PD1 nanobody that binds to such same or different epitope as the first PD1 nanobody; for example, contains a PD1 binding fragment and a CTLA4 binding fragment; a PD1 binding fragment and a BTLA binding fragment; a PD1 binding fragment and a LAG3 binding fragment; or a PD1-binding fragment, a LAG3-binding fragment, and a BTLA-binding fragment.

The present invention includes a polypeptide, ISVD or nanobody containing the amino acid sequence as described herein, for example, which is selected from SEQ ID NOs: 9-40.

The present invention also relates to a PD1 binding molecule of the present invention (eg, a single immunoglobulin variable domain (ISVD) or a multispecific ISVD such as a nanobody) in combination with an additional therapeutic agent.

The present invention provides injectable devices and vessels containing a PD1 binding molecule (eg, an immunoglobulin single variable domain (ISVD) or a multispecific ISVD such as a nanobody), optionally in combination with an additional therapeutic agent.

The present invention relates to a polynucleotide encoding a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD) or a polyspecific ISVD such as a nanobody), or a vector containing the polynucleotide, or a host cell containing the polynucleotide or vector.

The present invention relates to a method for producing a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD) or a multispecific ISVD, such as a nanobody), comprising introducing a polynucleotide encoding immunoglobulins into a host cell, and culturing the host cell in a medium under favorable conditions. for expressing said immunoglobulin from said polynucleotide; and optionally purifying the immunoglobulin from said host cell and/or said medium. PD1 binding molecules (e.g. ISVDs such as nano

- 4 041987 bodies) obtained 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 binding molecule (e.g., an immunoglobulin single variable domain (ISVD) or a multispecific ISVD such as a nanobody), optionally, in combination with an additional therapeutic agent.

The present invention also relates to a method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of a PD1 binding molecule of the present invention (e.g., an immunoglobulin single variable domain (ISVD) or a multispecific ISVD such as a nanobody), optionally in combination with an additional therapeutic agent. In addition, the present invention also relates to a method for the treatment or prevention of cancer (e.g., metastatic cancer, solid tumor, hematological cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, bladder transitional cell cancer, bladder cancer, tumor Wilma, 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 carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neoectodermal tumor, medulloblastoma, astrogliomas ependymomas, papillo we have a choroid plexus, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid or liver cancer, breast cancer, or stomach cancer) or an infectious disease (bacterial infection, viral infection, or fungal infection) in the subject's body, comprising administering to the subject an effective amount of a PD1 binding molecule (eg, an immunoglobulin single variable domain (ISVD) or a multispecific ISVD such as a nanobody), optionally in combination with an additional therapeutic agent. In an embodiment of the invention, the subject is also administered an additional therapeutic agent or an additional therapeutic procedure in combination with a PD1 binding molecule.

The present invention provides PD1 binding molecules (e.g., ISVDs such as nanobodies) that bind to PD1 containing: a CDR1 containing the amino acid sequence of IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6), a CDR2 containing the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V) or VITXSGGITY (SEQ ID NO: 7, where X is W or V), and a CDR3 containing the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F) that contain one or more mutations at position 11 (eg L11V) and 89 (eg I89L) or one or more mutations selected from E1D, L11V, a14P, W52aV, N73 (Q, P or S), A74S, K83R, I89L, W100aF relative to SEQ ID NO: 1 or 2. In an embodiment, the mutations are E1D, L11V, A14P, K83R and I89L, or L11V, A14P, K83R and I89L. In an embodiment, 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, the PD1 binding molecule contains the amino acid sequence: DVQLVESGGG WQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57).

The present invention also relates to LAG3 binding molecules that bind to LAG3 containing a CDR1 containing the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65), a CDR2 containing the amino acid sequence AISESGGRTH (SEQ ID NO: 66), and a CDR3 containing the amino acid sequence TLLWWTSEYAPIKMYDY (SEQ ID NO: 67), for example, containing the amino acid sequence: EVQLVE SGGGWQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKMYD YWGQGTLVTV SS (SEQ ID NO: 64).

The PD1 and LAG3 binding molecules can be a single molecule, such as the PD1/LAG3 binding molecule that is part of the present invention, which binds to PD1 and LAG3 and contains: a PD1 binding fragment containing: a CDR1 containing the amino acid sequence IHAMG (SEQ) ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6), a CDR2 containing the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4 where X is W or V) or VITXSGGITY (SEQ ID NO: 7 where X is W or V), and a CDR3 containing the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); and a LAG3 binding fragment containing: CDR1 containing the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65), CDR2 containing the amino acid sequence AISESGGRTH (SEQ ID NO: 66), and CDR3 containing the amino acid sequence TLLWWTSEYAPIKMYDY (SEQ ID NO: 67), and optionally a half-life increasing moiety, e.g., wherein the PD1 binding molecule contains the amino acid sequence

- 5 041987

DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDKHQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57); and the LAG3 binding molecule contains the amino acid sequence

EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKMYD YWGQGTLVTV SS (SEQ ID NO: 64); and optionally a half-life increasing moiety. For example, in an embodiment of the invention, the PD1/LAG3 binding molecule contains fragments, for example, in the following order:

PD1 binding molecule 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 35GS (eg GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));

LAG3 binding molecule 11B09 or 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L);

a peptide linker such as 9GS, 20GS or 35GS (eg GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGS (SEQ ID NO: 70)); a half-life increasing fragment such as ALB11002; and optionally a C-terminal extension such as alanine;

or a PD1 binding molecule 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 35GS (eg GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGS (SEQ ID NO: 70));

PD1 binding molecule 102C12 (L11V, A14P, A74S, K83R, I89L) or 1PD102C12 (L11V, A14P, W52aV, N73X (eg N73P or N73Q or N73S), A74S, K83R, I89L, W100aF);

a peptide linker such as 9GS, 20GS or 35 GS (eg GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGS (SEQ ID NO: 70));

LAG3 binding molecule 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L);

a peptide linker such as 9GS, 20GS or 35 GS (eg GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGS (SEQ ID NO: 70));

LAG3 binding molecule 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L);

a peptide linker such as 9GS, 20GS or 35 GS (eg GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGS (SEQ ID NO: 70));

a half-life increasing fragment such as ALB11002; and optionally a C-terminal extension such as alanine.

The half-life increasing fragment in an embodiment of the invention is a human serum albumin (HSA) binding molecule, such as ALB11002. In an embodiment, the HSA binding molecule comprises a CDR1 containing the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60), a CDR2 containing the amino acid sequence SISGSGSDTL (SEQ ID NO: 61), and a CDR3 containing the amino acid sequence GGSLSR (SEQ ID NO: 62) , for example, contains the amino acid sequence EVQLVESGGG WQPGNSLRL SCAASGFTFS SFGMSWVRQA PGKGLEWVSS

ISGSGSDTLYADSVKGRFTI SRDNAKTTLY LQMNSLRPED TALYYCTIGG SLSRSSQGTL VTVSSA (SEQ ID NO: 59). Such half-life increasing moieties are themselves part of the present invention.

In an embodiment, the PD1 binding molecule, LAG3 binding molecule, HSA binding molecule, and/or PD1/LAG3 binding molecule are in the injection device or vessel, optionally in combination with an additional therapeutic agent. Such a device or vessel is part of the present invention.

The present invention also relates to a polynucleotide encoding any of the binding molecules described herein, as well as to any vector (for example, a plasmid vector) containing a polynucleotide, as well as to any host cell (for example, a CHO cell or a cell of fungi, such as Pichia, eg P. past or is), comprising a polynucleotide or a vector, eg ectopic or integrated into one or more chromosomes of host cells.

The present invention also relates to a method for producing any of the binding molecules described herein, comprising introducing a polynucleotide encoding a binding molecule into a host cell (for example, a CHO cell or a cell of fungi such as Pichia, for example P. pastoris) and culturing the host cell in an environment under conditions favorable for expression of said binding molecule from said polynucleotide; and optionally, purifying the binding molecule from said host cell and/or said medium. Optionally, the vector or polynucleotide is inserted into one or more chromosomes of the host cells. Any binding molecule obtained in this way is also part of the present invention.

The present invention also relates to a method for preventing PD1 from binding to PD-L1 and/or

- 6 041987

PD-L2 or to inhibit any PD1 activity, e.g., as described herein, comprising contacting said PD1 with a PD1 binding molecule of the present invention (e.g., a PD1/LAG3 binding molecule), optionally in combination with an additional therapeutic agent. The present invention also relates to a method for preventing LAG3 from binding to MHC class II or for inhibiting any PD1 activity, e.g., as described herein, comprising contacting said LAG3 with a LAG3 binding molecule of the present invention (e.g., a PD1/LAG3 binding molecule), optionally , in combination with an additional therapeutic agent. The present invention also relates to a method of enhancing an immune response in a subject (e.g., human) comprising administering to the subject an effective amount of a binding molecule of the present invention (e.g., a PD1/LAG3 binding molecule), optionally in combination with an additional therapeutic agent. The present invention also relates to a method for treating or preventing cancer (e.g., metastatic cancer, solid tumor, or hematological cancer) or infectious disease (e.g., bacterial infection, viral infection, or fungal infection) in a subject (e.g., human), comprising administering to the subject an effective an amount of a binding molecule of the present invention (eg, a PD1/LAG3 binding molecule), optionally in combination with an additional therapeutic agent. When administered to a subject, a PD1 and/or LAG3 binding molecule of the present invention (eg, a PD1/LAG3 binding molecule) is optionally administered in conjunction with an additional therapeutic agent or therapeutic procedure, eg, surgical removal of a tumor.

Description of figures

Fig. 1. A table listing some of the amino acid positions that are referenced in this document and their numbering according to some alternative numbering systems (such as Aho and IMGT).

Fig. 2(1-5). Sequences of PD1 binding molecules.

Fig. 3 (A, B). Sequence alignment 102C12 with SEQ ID NO: 9-40 (see WO 2008/071447, SEQ ID NO: 348).

Fig. 4. Predominant N-linked glycans in monoclonal antibodies produced by Chinese hamster ovary cells (N-linked glycans of CHO cells) and genetically modified yeast cells (N-linked glycans of genetically modified yeast): squares: Nacetylglucosamine (GlcNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

Fig. 5. Monovalent binding of human PD1-expressing CHO-Ki cells to molecules

F023700275 (1PD102C12(A14P,A74S,K83R)-FLAG3-HIS6) or F023700706 (1PD102C12 (L11V,A14P,A74S,K83R,I89L)-FLAG3-HIS6). GAM=goat anti-mouse secondary antibody, used to detect mouse antibodies that bind to the FLAG epitope; αFLAG+GAM=goat anti-mouse secondary antibody used in conjunction with a mouse antibody that binds to the FLAG epitope located at the C-terminus of the construct.

Fig. 6. Monovalent binding of 3A9 cells expressing human LAG3 to F0237611B09-FLAG-His6 or F023700842 (F0237611B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-FLAG3-HIS6 molecules.

Fig. 7(A, B). Binding (A) of CHO cells expressing human PD-1 with F023700931, F023700924, F023700933 or F023700962 molecules; or (B) 3A9 cells expressing rhesus monkey PD-1, F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or Cho cells), F023700933, F023700962, F023700678 (1PD102C12 (A14P, A73R) -3, K83R)-3 -ALB11002) or F023701127. ABH0074+GAM=goat anti-mouse antibody used in conjunction with murine ABH0074 antibody that binds to nanobody scaffolds.

Fig. 8 (A, B). Binding (A) of CHO cells expressing human LAG3 with F023700931, F023700924 or F023700962 molecules; or (B) Za9 cells expressing rhesus monkey LAG3 with F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962.

Fig. 9 (A-H). Blocking binding between (A) human PD-L1-Fc and human PD1-expressing CHO-Ki cells with F023700275 or F023700706 molecules; (B) between human PD-L2-FC and human PD1-expressing CHO-Ki cells, F023700275 or F023700706 molecules; (C) between human PD-L1-Fc and human PD1-expressing CHO-Ki cells F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962; (D) between human PD-L2-FC and human PD1-expressing CHO-Ki cells F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962; (E) between human PDL1-Fc and human PD1-expressing CHO-Ki cells, F023700931, F023700924, F023700933 or F023700962 molecules; (F) between human PD-L2-Fc and human PD1-expressing CHO-Ki cells, F023700931, F023700924, F023700933 or F023700962 molecules; (G) between human PD-L1-Fc and human PD1-expressing CHO-Ki cells, molecules

- 7 041987

F023700929 (1PD102C12(L11V,A14P,A74S,K83R,I89L)-HIS6),

F023701190 (1PD102C12(E1D,L11V,A14P,W52aV,A74S, K83R, I89L,W100aF)-HIS6),

F023701192 (1PD102C12(E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6) or

F023701193 (1PD102C12(E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6);

(H) between human PD-L2-Fc and human PD1-expressing CHO-K1 cells, F023700929 molecules (1PD102C12(L11V,A14P,A74S,K83R,I89L)-HIS6),

F023701190 (1PD102C12(E1D,L11V,A14P,W52aV,A74S,K83R,I89L,W100aF)-HIS6),

F023701192 (1PD102C12(E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6) or

F023701193 (1PD102C12(E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6).

US=dyed; hPD-L1 EC30=hPD-L1-Fc staining intensity that was titrated to give 30% of the maximum staining intensity that could be obtained; GAH=KO3be is an anti-human secondary antibody used to detect the Fc region of a human antibody from hPD-Ll-Fc.

Fig. 10 (A-C). Blocking the binding of human LAG3-FC to Daudi cells by molecules (A) F0237611B09-FLAG3-HIS6, F023700842-FLAG3-HIS6 or human LAG3-FC; (B) F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962, human LAG3-Fc; or (C) F023700924, F023700931 or F023700962 or human LAG3-FC. Sec op1y=secondary antibody (GAH/GAM) only.

Fig. 11 (A, B). Proximity Assay (Beta-Galactosidase Enzyme Complementary Assay System): with (A) F023700931; F023701016, F023701017, control nanobody (IRR00085, respiratory syncytial virus (RSV) binding molecule), F023700933 or F023700962; or with (B) F023700924; F023700969, F023700970, control nanobody, F023700933 or F023700962.

Fig. 12 (A-E). Activation of Jurkat T cells (expression of luciferase operably linked to the IL2 promoter) in the presence of HSA with (A) F023700706 or a control nanobody (IRR00043, two anti-lysozyme nanobodies linked by a 3563-linker, with C-terminal FLAG3-His6), (В) F023700931 (экспрессируемых клетками Pichia или СНО), F023700924 (экспрессируемых клетками Pichia или СНО), F023700933, F023700962 или контрольного нанотела (IRR00085 или IRR00087, RSVсвязывающая молекула), (С) F023700924 F023700969, F023700970 или контрольного нанотела (IRR00043), (D) F023700931, F023701016, F023701017 или контрольного нанотела (IRR00043), или (Е) F023700706, F023701192 (1PD102C12(E1D,L11V,А14Р,W52aV,N73Q, A74S,K83R,I89L,W100aF)-HIS6 или F023701193 (1PD102C12 ( E1D,L11V,A14P,W52aV, N73P,A74S,K83R,I89L,W100aF)-HIS6 or control nanobody (IRR00088; RSV binding molecule).

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) using F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933, F023700962 or control nanobody; or from donors (M) 91, (N) 985, (O) 907 using F023700931, F023700924 or a control nanobody; or from donors (P) 91, (Q) 985 or (R) 907 using F023700924, F023700969, F023700970.

Fig. 14(A-F). Analysis using a mixed population of lymphocytes with CD4 T cells and dendritic cells from various donors, which determines (A-C) the production of interferon-gamma at various concentrations of nanobody F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia cells or CHO) or F023700933 or control antibody; (D-F) Interferon-gamma production at different concentrations of F023700924, F023700969, F023700970, F023700931 F023701016 or F023701017 nanobody or control nanobody.

Fig. 15(A-D). Analysis of activation of 3A9 T cells expressing human LAG3 in the presence of HSA and in the presence. (A) F023700656 (11B09 (E1D)), F023700842 or control IgG4; (B) F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962, or control nanobody (IRR00085 or IRR00087, RSV binding molecules); (C) F023700924, F023700969, F023700970 or control nanobody; (D) F023700931, F023701016 or F023701017, or control nanobody.

Fig. 16(A, B). Activation of Jurkat T cells expressing human LAG3 and human PD1 and antigen presenting Raji cells in the presence of (A) F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700862 (should be F023700892 ) or control nanobody; or (B) F023700924, F023700969, F023700970, F023700931, F023701016, F023701017, F023700933 or F023700962, or a control nanobody.

Fig. 17(A-C). Activation of a human T cell clone (interferon-gamma production) in the presence of JY cells expressing human PDL1 in the presence of varying concentrations of (A) F023700931 (expressed by Pichia or CHO cells), F023700924 (expressed by Pichia or CHO cells), F023700933 or F023700962, or a control nanobody; (B) F023700924, F023700969, F023700970, F023700933 or F023700962, or control nanobody; or (C) F023700931, F023701016, F023701017, F023700933 or F023700962, or a control nanobody.

- 8 041987

Fig. 18 (A-P). Sequences of the present invention.

Fig. 19(A-I). Reactivity of serum pre-existing antibodies (preAb) against F023700924 and F023700931 and the trivalent control nanobody T013700112 (which lacks mutations to reduce the binding of pre-existing antibodies) in the serum of healthy people (A) after 125 s and (B) 360 s; in the serum of people with cancer after (C) 125 s or (D) 360 s; or in the serum of patients suffering from (E) melanoma, (F) non-small cell lung cancer (NSCLC), (G) head and neck cancer, (h) gastric cancer, or (I) colorectal cancer.

Detailed description of the invention

The present invention relates to ISVDs that contain mutations that block the reactivity of pre-existing antibodies (preantibodies) to neoepitopes in the ISVD. Neoepitopes are epitopes in a protein that appear when the protein is mutated (eg shortened) or folded. Pre-existing antibodies are antibodies that exist in the patient's body prior to the administration of ISVD. The ISVDs of the present invention are based in part on lam antibodies whose C-terminal constant domains have been removed, thus exposing neoepitopes at the C-terminus of the resulting VHH for pre-antibody binding. It has been found that the combination of mutations at residues 11 and 89 (eg L11V and I89L or V89L) results in an unexpected lack of pre-antibody binding. Mutations at residue 112 have also been shown to remarkably reduce preantibody binding. Buyse & Button (Buyse & Boutton, WO2015/173325) included data showing that the combination of L11V and V89L mutations significantly reduced preantibody binding compared to L11V or V89L mutation alone. For example, in table. On page 97, Buses and Button compare data for an ISVD with a single V89L mutation (with or without C-terminal extension) versus the same ISVD with a V89L mutation plus an L11V mutation (again, with or without C-terminal extension). end). In addition, despite the fact that they were obtained in two separate experiments, the data shown in table. H for the L11V/V89L combination, compared to the data shown in Table B for the L11V mutation alone (in the same ISVD), showed that the reduction in pre-antibody binding obtained with the L11V/V89L combination was higher than for the mutation alone L11V. Because the llama antibody backbone structure is known to be highly conserved, the effect of mutations at positions 11 and 89 is likely to exist in any ISVD. Indeed, in FIG. 19 the effect was demonstrated for the current binding molecules, F023700924 and F023700931, which have been shown to have very low levels of preantibody binding in healthy individuals and in subjects suffering from cancer.

In the present invention, amino acid residues/positions in the variable domain of an immunoglobulin heavy chain will be numbered according to the Kabat system. For convenience, in Fig. 1 is a table listing some of the amino acid positions that are referenced in this document and their numbering according to some alternative numbering systems (such as Aho and IMGT). This point is also further discussed in the present description.

With regard to CDR, as is well known in the art, there are many ways to define and describe a CDR in 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 based on the location of structural loop regions). For example, see the website www.bioinf. or g.uk/abs/. In the present specification and claims, even if Kabat CDRs can be specified, it is most preferable to determine CDRs based on the Abm definition (which is based on Oxf or d Molecular's AbM antibody modeling software) as it is considered to be an optimal compromise between the Kabat and Chothia definitions. . Again see www.bioinf.or id.uk/abs/. See Sequences of Proteins of Immunological Interest, Rabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. no. 91-3242 (1991); Rabat (1978) Adv. Prot. Chem. 32:1-75; Rabat, et al., (1977) J. Biol. Chem. 252:6609-6616; 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. Rabat, Tai Te Wu, Carl Foeller, Harold M. Perry, Ray S. Gottesman (1991) Sequences of Proteins of Immunological Interest; Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Rontermann, R., Springer-Verlag, Heidelberg). In an embodiment of the invention, the definition of CDR is given according to Kabat, for example, where FR1 VHH contains amino acid residues at positions 1-30, CDR1 VHH contains amino acid residues at positions 31-35, FR2 VHH contains amino acids at positions 36-49, CDR2 VHH contains amino acid residues at positions 50-65, FR3 VHH contains amino acid residues at positions 66-94, CDR3 VHH contains amino acid residues at positions 95-102, and FR4 VHH contains amino acid residues at positions 103-113.

In an embodiment of the invention, CDRs are determined by Kontermann and Dubel (Rontermann and Dubel, Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010).

The term immunoglobulin single variable domain (also called ISV or ISVD) is commonly used to describe immunoglobulin variable domains (which can 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 the VH/VL interaction that is required of the VH and VL domains of the conventional 4-strand

- 9 041987 monoclonal antibody). Examples of ISVDs will be apparent to the skilled artisan and, for example, include nanobodies (including VHH, humanized VHH and/or VH modified for camelid antibodies, such as modified for human camelid VH antibodies), IgNARs, domains, (single domain) antibodies ( such as dAb™) which are VH domains or which are derived from VH domains, and (single domain) antibodies (such as dAb™) which are VL domains or which are derived from VL domains. ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, the ISVD is a nanobody.

The term nanobody is generally defined in WO 08/020079 or WO 09/138519, and thus, in a specific aspect, it generally means VHH, humanized VHH, or modified for camelid VH antibodies (such as modified for human camelid VH antibodies). ) or, more generally, an optimized VHH sequence (eg, optimized for chemical stability and/or solubility, maximum overlap with known human antibody frameworks, and maximum expression). It should be noted that the terms nanobody or nanobodies are registered trademarks of Ablynx N.V and may therefore also be referred to as Nanobody® and/or Nanobody®). An example of ISVD is 102C12 (E1D, L11V, A14P, A74S, K83R, I89L). Other ISVDs are also presented in Table. A and C in this document.

A multispecific binding molecule (e.g., multispecific ISVD) is a molecule that contains, for example, a first PD1 or LAG3 binding moiety (e.g., ISVD or nanobody) and one or more (e.g., 1, 2, 3, 4, 5) additional binding fragments (eg ISVD or nanobodies) that bind to an epitope different from that of the first PD1 or LAG3 binding fragment (eg CTLA4, CD27 and/or BTLA); for example, contains a PD1 or LAG3 binding fragment and a CTLA4 binding fragment, a PD1 or LAG3 binding fragment and a BTLA binding fragment, one or two PD1 binding fragments and one or two LAG3 binding fragments and a human albumin binding fragment, or a PD1 binding fragment, a LAG3 binding fragment and a BTLA binding fragment. The polyspecific binding molecule is, for example, F023700931 or F023700899.

A binding moiety or binding domain or binding unit is a molecule, such as an ISVD or nanobody, that binds to an antigen. The binding fragment or binding domain or binding unit may be part of a larger molecule, such as a polyvalent or multispecific immunoglobulin, which includes more than one fragment, domain, or unit, and/or which contains another functional element, such as, for example, a fragment that increases the half-life (HLE), targeting molecule and/or small molecule compound such as polyethylene glycol (PEG).

A monovalent PD1 or LAG3 binding molecule (eg, ISVD, such as a nanobody) is a molecule that contains a single antigen binding domain. A bivalent PD1 or LAG3 binding molecule contains two antigen binding domains (eg, conventional antibodies, including bispecific antibodies). A multivalent PD1 or LAG3 binding molecule contains more than one antigen binding domain.

A monospecific PD1 or LAG3 binding molecule binds one antigen, a bispecific PD1 or LAG3 binding molecule binds to two different antigens, and a polyspecific PD1 or LAG3 binding molecule binds to more than one antigen.

The biparatopic PD1 or LAG3 binding molecule is monospecific but binds to two different epitopes on the same antigen. A polyparatopic PD1 or LAG3 binding molecule binds the same antigen, but more than one epitope in the antigen.

As used herein, the term half-life referring to a PD1 and/or LAG3 binding molecule or ISVD, a nanobody, an ISVD-based biocompound, a nanobody-based biocompound, or any other polypeptide can be defined as described in point (o) on page 57 in WO08/020079 and as above refers to the time taken for the serum concentration of the polypeptide to decrease by 50% in vivo, for example due to degradation of the polypeptide and/or clearance or sequestration of the polypeptide by natural mechanisms. The in vivo half-life of a polypeptide of the invention can be determined by any known method such as pharmacokinetic analysis. Suitable methods will be understood by a person skilled in the art and in the General case may, for example, be as described in paragraph (o) on page 57 in WO 08/020079. As also stated in paragraph (o) on page 57 in WO 08/020079, the half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the curve (AUC). In this regard, it should be noted that the term half-life as used herein specifically refers to the t1/2-beta half-life or terminal half-life (where t1/2alpha and/or AUC, or both, may be ignored). ). For example, see the Experimental section (below), as well as standard references such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach

- 10 041987 (1996). See also Pharmacokinetics, M Gibaldi & D Perron published by Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms increased half-life or increased half-life, as also defined in point (o) on page 57 of WO 08/020079 specifically refer to an increase in t1/2-beta, either with or without an increase in t1 /2-alpha and/or AUC, or both.

When the definition of a term is not expressly given in this document, this term will have the meaning usual in the art, which will be understood by a person skilled in the art. For example, reference may be made to standard references such as Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York (1987); Lewin, Genes II, John Wiley & Sons, New York, N.Y., (1985); Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., Immunology (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., Immunobiology (6th Ed.), Garl and Science Publishing/Churchill Livingstone, New Wilick (2005), and the background referenced herein.

For a general description of multivalent and multispecific polypeptides containing one or more nanobodies and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; and also, for example, patents WO 96/34103, WO 99/23221, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

Isolated PD1 and/or LAG3 binding molecules (eg, ISVDs such as nanobodies), polypeptides, polynucleotides, and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are derived. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cell debris and growth media. Isolated PD1 and/or LAG3 binding molecules may additionally be at least partially free of components of the expression system, such as biological molecules from the host cell or its growth medium. In general, the term isolated is not intended to mean the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical composition that includes antibodies or fragments.

The phrase control sequences refers to polynucleotides necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Promoters, polyadenylation signals and enhancers are known to be used in the case of eukaryotic cells.

A nucleic acid or polynucleotide is operably linked when it is in operable interaction with another polynucleotide. For example, a DNA sequence of a precursor or secreted leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that is involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if they affect the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is located to allow translation. In general, but not always, operably linked means that the linked DNA sequences are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to common practice. The present invention includes polynucleotides encoding PD1 and/or LAG3 binding molecules that are optionally operably linked to one or more control sequences such as a promoter.

A PD1 and/or LAG3 binding molecule refers to a binding molecule that includes a PD1 binding moiety, or a LAG3 binding moiety, or both a PD1 binding moiety and a LAG3 binding moiety (i.e., a PD1/LAG3 binding moiety) and optionally another binding moiety, which binds, for example, human serum albumin. In an embodiment, the PD1/LAG binding molecule is

102С12 (E1D,L11V, A14P, A74S, K83R, I89L)-35GS-11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A; or

102С12 (E1D,L11V,A14P,A74S,K83R,I89L)-35GS-102C12 (L11V,A14P,A74S,K83R,I89L)-35GS-11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L) -35GS-11B09

- 11 041987 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A;

given in the present description.

See F023700924 or F023700931. The PD1/LAG3 binding molecule includes a PD1 binding moiety and a LAG3 binding moiety.

Any PD1 binding molecule shown in FIG. 18(A-P) (or any PD1 binding molecule containing the CDR1, CDR2, and CDR3 of such a PD1 binding molecule), any LAG3 binding molecule shown in FIG. 18 (A-P) (or any LAG3 binding molecule containing the CDR1, CDR2 and CDR3 of such LAG3 binding molecule) or any PD1/LAG3 binding molecule shown in FIG. 18 (AP), or any PD1/LAG3 binding molecule containing the CDR1, CDR2 and CDR3 of its PD1 binding fragment and/or its LAG3 binding fragment. The binding molecules shown in FIG. 18 (A-P), in an embodiment, do not include C-terminal extension (e.g. A), FLAG and/or HIS tags (e.g. HHHHHH (amino acids 29-34 or SEQ ID NO: 68), AAAHNNHHHH (SEQ ID NO: 69) or AAADYKDHDGDYKDHDIDYKDDDKGAAHHHHH (SEQ ID NO: 68)). Any such binding molecule or CDR may, in an embodiment, be a variant of the sequence shown in FIG. 18 (A-P), for example, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations (eg, conservative substitutions or deletions).

In general, the basic structural unit of an antibody comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, with each pair including one light (about 25 kDa) and one heavy (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal region of the heavy chain refers to a constant region mainly responsible for effector function. Generally, human antibody light chains are classified into kappa and lambda light chains. In addition, the heavy chains of human antibodies are generally classified into mu, delta, gamma, alpha, or epsilon and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a J region of about 12 amino acids, with the heavy chain also including a D region of about 10 or more amino acids. In general, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

Examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments and single chain Fv molecules.

PD1 binding molecule or PD1 ISVD or PD1 nanobody refers to a binding molecule or ISVD or nanobody, respectively, that binds to PD1. A similar definition can be applied to molecules that bind to LAG3 or CTLA4 or another antigen.

The following properties are associated with these mutations in the PD1 binding molecule, 102C12:

E1D: prevents the formation of pyroglutamic acid in the first amino acid of construct E1

L11V: reduces pre-antibody binding

A14P: humanization

W52aV: : warns ¹ oxidation W52a N73P: warns deamidation N73 N73Q: warns deamidation N73 N73S: warns deamidation N73 A74S: humanization K83R: humanization I89L: reduces preantibody binding

WIOOaF: prevents oxidation of W100a

Or in the LAG3 binding molecule, PVO9

L11V: reduces pre-antibody binding

A14P: humanization

- 12 041987

R41P: humanization

N43K: humanization

A62S: humanization

A74S: humanization

K83R: humanization

V8 9L: Reduces pre-antibody binding

In an embodiment of the invention, PD1 is human PD1. In an embodiment of the invention, human PD1 contains the amino acid sequence:

MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLWTEGDNA

TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL

PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE

VPTAHPSPSP RPAGQFQTLV VGWGGLLGS LVLLVWVLAV ICSRAARGTI

GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVVP CVPEQTEYAT

IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 137).

In an embodiment, the LAG3 is human LAG3. In the osu- According to the invention, human LAG3 contains the amino acid sequence: MWEAQFLGL QDLSLLRRAG FLQPLWVAPV KPLQPGAEVP WWAQEGAPA QLPCSPTIPL VTWQHQPDSG SGRLPLQPRV PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLR QLDERGRQRG MTASPPGSLR DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQAS ASDWVILNCS VSPMDSGPWG FSRPDRPASV HWFRRNRGQGR VPVRESPHHH LAESFLFLPQ CILTYRDGFN RSFLTAKWTP VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT PGGGPDLLVT ITVTPKSFGS GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI PGSLGKLLCE QCQLYQGERL VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQPW LGAAVYFTEL FHLWRRQWRP SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFG

RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL (SEQ ID NO: 138)

PD1 binding molecules.

The present invention relates to improved PD1 binding molecules, for example improved PD1-ISVDs, and in particular improved PD1 nanobodies. Improved PD1 binding molecules of the invention are also referred to herein as PD1 binding molecules of the invention or PD1 binding molecules.

The monovalent PD1 binding molecules of the present invention discussed herein will comprise the amino acid sequence of SEQ ID NO: 1 or 2, but include one or more mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 or 112 (or at any of the mutation positions provided herein for the PD1 binding molecules of the invention).

Multispecific PD1 binding molecules discussed herein, for example, which include LAG3, will include PD1 binding fragments, including CDR1, CDR2, and CDR3, which are present in the PD1 binding molecules shown in Table 1 below. A-1 and A-2 (for example, in 102C12 or in reference sequence A). Optionally, the PD1-binding fragment of the polyspe

- 13 041987 cific binding molecule contains the amino acids from SEQ ID NO: 1 or 2, but includes one or more mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 or 112.

The present invention relates to improved PD1 binding molecules, eg improved PD1-ISVDs, and in particular improved PD1 nanobodies. PD1 binding molecules of the present invention include polypeptides that are variants of polypeptides containing the amino acid sequence of SEQ ID NO: 1 or 2, which is mutated at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/ or 112. Improved PD1 binding molecules of the invention are also referred to herein as PD1 binding molecules of the invention or PD1 binding molecules. These terms encompass any molecule that binds to PD1 and includes any of the molecules that bind to PD1 and are provided herein. For example, the terms include an ISVD that contains the amino acid sequence listed in a member selected from the group consisting of SEQ ID NOs: 9-40 and 57, as well as any conventional antibody or antigen-binding fragment thereof that includes the amino acid sequence listed in the sequences selected from the group consisting of SEQ ID NOs: 9-40 and 57; a multispecific immunoglobulin, such as a bispecific immunoglobulin (e.g., ISVD) that contains the amino acid sequence specified in a member selected from the group consisting of SEQ ID NOs: 9-40 and 57, binds to PD1 and also binds to another antigen, such as other epitope PD1, CD27, LAG3, CTLA4, BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1, PD-L2, ILT1, ILT2 CEACAM1, CEACAM5, TIM3, TIGIT, VISTA, ILT3 , ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40, CD137, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17, TSLP , for example, containing a PD1-binding fragment and a CTLA4-binding fragment; a PD1 binding fragment and a BTLA binding fragment; a PD1 binding fragment and a LAG3 binding fragment; or a PD1-binding fragment, a LAG3-binding fragment, and a BTLA-binding fragment. A multivalent PD1 binding molecule contains more than one antigen binding domain, eg more than one ISVD (one or more of which binds to PD1). A monospecific PD1 binding molecule binds one antigen (PD1); a bispecific PD1 binding molecule binds to two different antigens (one of which is PD1), and a multispecific PD binding molecule binds to more than one antigen (one or more of which is PD1 and one or more of which is another antigen) . For example, in an embodiment, the PD1 binding molecule comprises an amino acid sequence selected from SEQ ID NOs: 106-124.

More specifically, the invention is directed to improved PD1 binding molecules that are variants of 102C12 and the A reference sequence and that bind less strongly to undesirable factors (commonly referred to as pre-existing antibodies) that may be present in the sera of some healthy individuals as well as diseased individuals. See WO 12/175741, WO 2013/024059 and also, for example, Holl and et al. (J. Clin. Immunol., 2013, 33(7):1192-203), as well as PCT application PCT/EP2015/060643 (WO2015/173325).

As further described herein, the PD1 binding molecules of the invention preferably have the same combination of CDRs (i.e., CDR1, CDR2, and CDR3) that are present in 102C12 or reference sequence A. WO 2008/071447 describes nanobodies that can bind to PD1, and their application. SEQ ID NO: 348 of WO 2008/071447 discloses a PD-1 specific nanobody referred to as 102C12, the sequence of which is given herein as SEQ ID NO: 1. In addition, a variant of 102C12 with the humanizing substitution Q108L (also referred to herein as reference sequence A) is used in the present invention as a reference compound, and its sequence is given herein as SEQ ID NO: 2. The present invention includes PD1 binding molecules that include a mutation at position 108, for example Q108L.

The present invention includes PD1-binding molecules that are 102C12 variants and PD1/LAG3-binding molecules that contain those 102C12 variants as shown in Table 1 below. A-2. The scope of the present invention includes binding PD1 molecules, which include the CDR1, CDR2 and CDR3 of these options, shown below in table. A-2, as well as PD1/LAG3-binding molecules containing a PD1-binding fragment that includes CDR1, CDR2 and CDR3 of the indicated variants shown in Table 1 below. A-2.

- 14 041987

Table A-1

PD1 binding molecule, 102C12

SEQ ID NO Description Subsequence 1 WO 2008/071447, SEQ ID NO: 348 (102C12) (may be specified in this document as "1PD102C12") EVOLVESGGGLVOAGGSLRLSCAASGSIASIHAMGWFROA PGKE RE FVAVITWSGGITYYADSVKGRFTISRDNAKNTVY LOMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTQVTVSS 2 reference sequence A EVOLVESGGGLVOAGGSLRLSCAASGSIASIHAMGWFRQA PGKE RE FVAVITWSGGITYYADSVKGRFTISRDNAKNTVY LOMNSLKPEDTAIYYCAGDKHQSSWYDYWGOGTLVTVSS

Table A-2

PD1 binding molecules that are sequence-optimized 102C12 variants (optionally fused to HSA binding moieties)

Monomeric SO 1PD102C12, 102C12 (EID, L11V, A14P, A74S, K83R, I89L) Target : hPD-1 SEQ ID NO: 57 DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS SEQ ID NO: 3 CDR1 of the PD1 binding fragment (no Kabatu) IHAMG SEQ ID NO: 4 CDR2 of the PD1 binding fragment (according to Kabat) VITXSGGITYYADSVKG where X is W or V (for example, VITwSGGITYYADSVKG or VITvSGGITYYADSVKG) SEQ ID NO: 5 CDR3 binding PD1 fragment (according to Kabatu/Abm) DKHQSSXYDY where X is W or F (e.g. DKHQSSwYDY or DKHQSSfYDY) SEQ ID NO: 6 CDR1 of PD1 binding fragment (Abm) GS IAS IHAMG SEQ ID NO: 7 CDR2 of PD1 binding fragment (Abm) VITXSGGITY where X is W or V (e.g. VITwSGGITY or VITvSGGITY) SEQ ID NO: 8 CDR3 binding PD1 fragment (by Kabat/Abm) DKHQSSXYDY where X is W or F (e.g. DKHQSSwYDY or DKHQSSfYDY) Name: F023700275 Description: 1PD102C12 (A14P,A74S, K83R) Target: hPD-1 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL RPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS Monomeric SO 1PD102C12 Name: F023700706 or EVOLVESGGGVVOPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL

- 15 041987

F023700929 Description: 1PD102C12 (LIIV,A14R,A74S,K83R,I89L) Target : hPD-1 SEQ ID NO: 99 RPEDTALYYCAGDKHQSSWYDYWGOGTLVTVSS Monovalent SO 1PD102C12+ALB11002 Name: F023701127 Description: 1PD102C12 (EID,LIIV,A14P,A74S,K8 3R, I 89L)-35GS-ALB11002-A Target : hPD-1 SEQ ID NO: 101 DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL RPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSL RLSCAASGFT FSS FGMSWVRQAPGKGLEWVS SISGSGSDTLYA DSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSR SSQGTLVTVSSA Bivalent SO 1PD102C12+ALB11002 Name: F023700933 Description: 1PD102C12 (E1D,L11V,A14P,A74S,K83R,I 89L)-35GS1PD102C12(LIIV,A14P,A74S, K 83R,I89L)-35GS-ALB11002-A Target : hPD-1 SEQ ID NO: 102 DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL RPEDTALYYCAGDKHQSSWYDYWGOGTLVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSL RL S CAAS GSIASIHAMGWFRQAPGKERE FVAVITWSGGITY Y A DSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSS WYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Improved monomeric SO 1PD102C12 Name: F023701190 Description: 1PD102C12(EID,L11V,A14P,W5 2aV,A74S,K83R,189L,WIOOaF) Target : hPD-1 SEQ ID NO: 103 Optionally, F023701190 contains an N73X mutation such as N73S DVQLVESGGGVVOPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL RPEDTALYYCAGDKHQSSFYDYWGOGTLVTVSS Improved Monomeric SO 1PD102C12 Name: F023701192 Description: 1PD102C12(EID,L11V,A14P,W5 2aV,N73Q,A74S,K83R,I89L,W1 OOaF) Target : hPD-1 SEQ ID NO: 104 DVQLVESGGGVVOPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSL RPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS Improved Monomeric SO 1PD102C12 Name: F023701193 Description: 1PD102C12 (EID,LIIV,A14P,W52aV,N73P,A74S,K83R,189L,WIOOaF) Target : hPD-1 SEQ ID NO: 105 DVOLVESGGGVVOPGGSLRLSCAASGSIASIHAMGWFROAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLOMNSL RPEDTALYYCAGDKHQSSFYDYWGOGTLVTVSS F023700641 Description : 1PD102C12(A14P,A74S,K83R)- 35GS- 1PD102C12(A14P,A74S,K83R)- 35GS-ALB11002 Target : hPD-1 EVQLVESGGGLVOPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL RPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL RL S CAAS GSIASIHAMGWFRQAPGKERE FVAVITWSGGITYYA DSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSS WYDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG

- 16 041987

SEQ ID NO: 126 GSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 1PD102C12 (EID, L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF) amino acids 1-19 from SEQ ID NO: 113 DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSL RPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS 1PD102C12 (EID, L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WIOOaF) amino acids 1-19 from SEQ ID NO: 117 DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDOSKNTVYLOMNSL RPEDTALYYCAGDKHQSSFYDYWGOGTLVTVSS 1PD102C12 (EID, L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WIOOaF) amino acids 1-19 from SEQ ID NO: 121 DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGK ERE FVAVITVSGGITYYADSVKGRFTISRDS SKNTVYLQMNSL RPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS

*CDRs of PD1 binding molecules are underlined and/or in bold

The present invention includes embodiments in which the PD1 binding molecule contains one, two, or three CDRs that are present in the PD1 binding molecule, which is listed above in table. A-1 or A-2 (e.g., SEQ ID NO: 57, 98, 99, 103, 104, or 105), but which include 0, 1, 2, 3, 4, or 5 amino acid substitutions, such as conservative substitutions, and /or have 100, 99, 98, 97, 96 or 95% sequence identity with the CDRs listed in table. A-1 or A-2, wherein the PD1 binding molecule having such CDRs retains the ability to bind to PD1. In an embodiment, the first amino acid of a PD1 binding molecule of the present invention is E. In an embodiment, the first amino acid of a PD1 binding molecule of the present invention is D. PD1/LAG3 binding molecules comprising such a PD1/LAG3 binding fragment are part of the present invention .

The present invention includes any PD1 binding molecule containing the amino acid sequence of SEQ ID NO: 57, 98, 99, 103, 104, or 105 or an amino acid sequence having 80% or more (e.g., 85%, 90%, 95% 96%, 97 %, 98%, or 99%) amino acid sequence identity (i.e. when comparing full length amino acid sequences), where the PD1 binding molecule retains the ability to bind to PD1, and optionally includes an HSA binding moiety. PD1/LAG3 binding molecules containing such a PD1/LAG3 binding moiety are part of the present invention.

A PD1 binding molecule described as 102C12 (E1D, L11V, A14P, A74S, K83R, I89L) refers to a binding molecule having the sequence 102C12 (SEQ ID NO: 1) or reference sequence A (SEQ ID NO: 2), but where the sequence contains mutations E1D, L11V, A14P, A74S, K83R and I89L. This note is used throughout the description in relation to several different binding molecules. For example, 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) refers to a LAG3 binding molecule containing the amino acid sequence 11B09 (SEQ ID NO: 64), but where the sequence includes the mutations L11V, A14P, R41P, N43K , A62S, A74S, K83R and V89L.

Residue numbers according to the Kabat system for some of the residues of the PD1-binding molecules listed in Table 1. A, shown in sequence below:

E iVQLVE S GGGL ₁₁ V ₁₂ Q ₁₃ A ₁₄ GGS LRL S CAAS G ₂₆ S ₂₇ 128A29S301HAMGW ₃ 6F3 ₇ R ₃₈ Q3 ₉ AP _{4 1 GKERE 46} F ₄₇ V ₄₈ A ₄₉ VITW5 _2a SGGITYYADSVKGR ₆₆ F ₆₉ T S ₆₈ I ₆₈ oRDN ₇₃ A ₇₄ KNTVYLQM ₈₂ N _82a S

82bL82cK83P84EDT ₈₇ A ₈₈ I ₈₉ Y ₉ oY ₉ iCAGDKHQSSW ₁ ooaYDYWio3Gio ₄ Qio5Gio6Tio7Lio8Vio9TiioVi

11S112S113 (SEQ ID NO: 2) .

The Kabat residue numbers for some 102C12 residues (E1D, L11V, A14P, A74S, K83R, I89L) are shown in the sequence below:

D1VQLVESGGG V11VQP14GGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNS74KNTVY LQMNSLR ₈₃ PED TAL89YYCAGDK HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57). Optionally, residue 1 is E.

- 17 041987

Mutations may be referred to herein and are designated by Kabat numbers as shown above.

Some preferred, but not limited, PD1 binding molecules of the invention are 102C12 (E1D (optional), L11V, A14P, A74S, K83R, I89L) containing the amino acid sequence shown in SEQ ID NOs: 9-40, 57, 98, 99 or 101-105, or shown in FIG. 2, fig. 3 or fig. 18 (A-P). PD1 binding molecules from SEQ ID NOs: 24-40, 101 or 102 are examples of PD1 binding molecules of the invention having an alanine C-terminus extension, i.e. an alanine residue at the C-terminus of the ISVD sequence (also sometimes referred to as position 114) compared to conventional C-terminal sequence VTVSS (SEQ ID NO: 52 present in reference sequence A). This C-terminal extension with alanine may prevent so-called pre-existing antibodies (presumably IgG) from binding to a putative epitope that is located in the C-terminal region of the ISV. This epitope is expected to include, among other residues, the surface-exposed amino acid residues of the C-terminal sequence of VTVSS (SEQ ID NO: 52) as well as the amino acid residue at position 14 (and the amino acid residues adjacent/near it in the amino acid sequence, such as positions 11, 13, and 15), and may contain an amino acid residue at position 83 (and amino acid residues adjacent/near it in the amino acid sequence, such as positions 82, 82a, 82b, and 84), and/or an amino acid residue at position 108 (and amino acid residues adjacent/near it in the amino acid sequence, such as position 107).

However, while the presence of such a C-terminal alanine (or, in general, a C-terminal extension) can significantly reduce (or, in some cases, essentially completely prevent) binding by pre-existing antibodies that can be found in the sera of a number of subjects (as healthy subjects and subjects with pathologies or diseases), it has been found that the sera of some subjects (such as sera from patients with certain immune diseases such as SLE) may contain pre-existing antibodies that can bind to the C-terminal region of ISV (when such area exposed), even when the ISV contains such a C-terminal alanine (or, more broadly, such a C-terminal extension).

Accordingly, one particular object of the invention is to provide PD1 binding molecules which are improved versions of the PD1 nanobody, herein referred to as reference sequence A, and which have reduced binding by so-called pre-existing antibodies and in particular by the type of antibodies described in PCT/EP 2015/060643 (WO2015/173325) (i.e. such pre-existing antibodies that can bind to the exposed C-terminal region of the ISV even when the C-terminus is extended).

The invention provides PD1 binding molecules comprising amino acid sequences that are variants of SEQ ID NO: 1 or 2 that contain one or more of the following mutations compared to SEQ ID NO: 1 or 2:

1D or 1E;

11V;

14P;

52aV

73Q, 73P or 73S

74S;

83R;

89T or 89L;

100aF

1D or 1E in combination with 11V, 14P, 74S, 83R and 89L;

1D in combination with 11V, 14P, 52aV, 73Q 73S or 73P, 74S, 83R, 89L and 100aF;

1E in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and 100aF;

89L in combination with 11V, 14P, 52aV, 73S, or 73Q, or 73P, 74S, 83R and 100aF, and optionally 1D;

89L in combination with 11V;

89L combined with 110K or 110Q;

89L combined with 112K or 112Q;

89L in combination with 11V, 14P, 74S, 83R, and optionally 1D;

110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and 100aF and optionally 1D;

112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and 100aF and optionally 1D;

89L in combination with 11V and 110K or 110Q;

89L in combination with 11V and 112K or 112Q;

11V combined with 110K or 110Q; or

11V combined with 112K or 112Q.

In particular, PD1 binding molecules (e.g. ISVDs such as nanobodies) proposed in

- 18 041987 invention, include a variant of SEQ ID NO: 1 or 2, where in the embodiment of the invention the amino acid at position 1 is selected from E and D;

the amino acid at position 11 is selected from L and V;

the amino acid at position 14 is selected from A and P;

the amino acid at position 52a is selected from W and V;

the amino acid at position 73 is selected from N, S, P and Q;

the amino acid at position 74 is selected from A or S;

the amino acid at position 83 is selected from K or R;

the amino acid at position 89 is selected from T, V, I, or L;

the amino acid at position 100a is selected from W and F;

the amino acid at position 110 is selected from T, K or Q; and/or the amino acid at position 112 is selected from S, K or Q;

for example, where the PD1 binding molecule contains one or more mutations:

(i) position 1 is D or E;

(ii) position 11 is V;

(iii) position 14 is P;

(iv) position 52a is V;

(v) position 73 is P, S or Q;

(vi) position 74 is S;

(vii) position 83 is R;

(viii) position 89 is T or L;

(ix) position 100a is F; for example, contains a set of mutations listed below:

a) position 1 is D or E, position 11 is V, position 14 is P, position 74 is S, position 83 is R, and position 89 is L;

b) position 1 is D or E, position 11 is V, position 14 is P, position 52a is V, position 73 is S, P or Q; position 74 is S, position 83 is R, position 89 is L, and position 100a is F;

c) position 89 is L and position 11 is V;

d) position 89 is L and position 110 is K or Q;

e) position 89 is L and position 112 is K or Q;

f) position 1 is D or E, position 11 is V, position 14 is P, position 52a is V, position 73 is S, P or Q; position 74 is S, position 83 is R; position 89 is L, position 100a is F, and position 110 is K or Q;

g) position 1 is D or E, position 11 is V, position 14 is P, position 52a is V, position 73 is S, P or Q, position 74 is S, position 83 is R; position 89 is L, position 100a is F, and position 112 is K or Q;

h) position 89 is L and position 11 is V and position 110 is K or Q;

i) position 89 is L and position 11 is V and position 112 is K or Q;

j) position 11 is V and position 110 is K or Q; and/or

k) position 11 is V and position 112 is K or Q; relative to the amino acid sequence of SEQ ID NO: 1 or 2.

In specific embodiments, PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention, comprising amino acid sequences that are variants of SEQ ID NO: 1 or SEQ ID NO: 2, in which position 89 is T or L, or in where position 1 is V, position 14 is P, position 74 is S, position 83 is R, and position 89 is L, or where position 11 is V and position 89 is L (optional in suitable combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation, and in particular in combination with a 110K or 110Q mutation) are also part of the present invention. The present invention includes amino acid sequences in which position 11 is V and position 89 is L, optionally with a 110K or 110Q mutation.

As already indicated, the PD1 binding molecules described in the present invention can bind (and, in particular, can specifically bind) to PD-1. In an embodiment of the invention, they can bind to PD1 and inhibit binding between PD1 and PD-L1 and/or PD-L2. For example, in an embodiment, the PD1 binding molecules of the present invention bind to PD-1 and free T cells from PD-1-mediated inhibition of T

- 19 041987 cellular immune response (for example, freeing T cells from PD1-mediated inhibition of proliferation and production of cytokines).

Table B lists some non-limiting possible combinations of amino acid residues that may be present at positions 11, 89, 110 and 112 in the PD1 binding molecules of the invention.

Table B

Possible combinations of mutations at amino acid positions 11, 89, 110 and 112 in variants of PD1 binding molecules of SEQ ID NO: 1 or 2

POSITION POSITION With about m in I N A T I O N eleven 89 By 112 with o m in I N A T I O N eleven 89 By 112 L T T S V T T S L T T To V T T To L T T Q V T T Q L T To S V T To S L T Q S V T Q S L V T To V V T To L V T Q V V T Q L V To S V V To S L V Q S V V Q S L I T To V I T To L I T Q V I T Q L I To S V I To S L I Q S V I Q S V L T S

These positions can be combined with mutations such as E1D, A14P, A74S and/or K83R (and/or others).

The PD1 binding molecules of the invention are further described in the text, examples and figures of this document, namely, they contain CDRs as described herein and have a general degree of sequence identity (as defined herein) with the sequences of SEQ ID NO: 1 or 2 that are disclosed herein and/or may have a limited number of amino acid differences (as described herein) with these reference sequences (one of them).

PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention that contain the amino acid sequence of SEQ ID NO: 1 or 2 and one or more mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a , 110 and/or 112 preferably include the following CDRs (in the Kabat system):

CDR1 (according to Kabat), which is the amino acid sequence of IKAMG (SEQ ID NO: 3); And

CDR2 (by Kabat), which is the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V); And

CDR3 (according to Kabat), which is the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); optionally, where CDR1, CDR2 and/or CDR3 have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, eg conservative substitutions.

Alternatively, when the CDRs are given according to Abm numbering, the PD1 binding molecules (e.g., ISVDs, such as nanobodies) of the invention preferably include the following CDRs:

CDR1 (according to Abm), which is the amino acid sequence of GSIASIHAMG (SEQ ID NO: 6); And

CDR2 (according to Abm), which is the amino acid sequence VITXSGGITY (SEQ ID NO: 7, where X is W or V); And

CDR3 (by Abm), which is the amino acid sequence DKHQSSXYDY (SEQ ID NO: 8, which is the same as SEQ ID NO: 5, where X is W or F); optionally, where CDR1, CDR2 and/or CDR3 have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, eg conservative substitutions.

The PD1 binding molecules (e.g. ISVDs such as nanobodies) of the invention, in addition to the CDR1, CDR2 and CDR3 set forth above, preferably also have a degree of identity in

- 20 041987 sequences with the amino acid sequence of SEQ ID NO: 1 or 2 at least 85%, preferably at least 90%, more preferably at least 95% (for example, 100%) (where CDR, any extension of C- end that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect, are not taken into account when determining the degree of sequence identity) when comparing is performed using the BLAST algorithm, in which the algorithm parameters are chosen to provide the greatest match between matching sequences over the entire length of the matching reference sequences (e.g. wait threshold: 10; word length: 3; maximum match in query range: 0; BLOSUM matrix 62 ; gap costs: existence 11, expansion 1; conditional compositional adjustment of the evaluation matrix); and/or no more than 7, e.g. no more than 5, preferably no more than 3, e.g. only 3, 2 or 1 amino acid differences with the amino acid sequence of SEQ ID NO: 1 or 2 (wherein said amino acid differences, if any, may present in framework regions and/or CDRs, but preferably present only in framework regions and not in CDRs, disregarding any C-terminal extension that may be present and disregarding mutations at positions 1, 11, 14, 52a , 73, 74, 83, 89, 100a, 110 and/or 112 required in accordance with a particular aspect).

The following references refer to BLAST algorithms that are often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J.M. et al. , (1994) Comput. Appl. biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., A model of evolutionary change in proteins. in Atlas of Protein Sequence and Structure, (1978) vol. 5 suppl. 3.M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., Matrices for detecting distant relationships. in Atlas of Protein Sequence and Structure, (1978) vol. 5 suppl. 3.M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. sci. USA 89:10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S.F. Evaluating the statistical significance of multiple distinct local alignments. in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

With respect to the various aspects and preferred aspects of the PD1 binding molecules (e.g. ISVDs such as nanobodies) of the invention, when it comes to the degree of overall sequence identity with respect to SEQ ID NO: 1 or 2 and/or the number and type of amino acids differences that may be present in such a binding molecule of the present invention (namely, in comparison with the sequences of SEQ ID NO: 1 or 2), it should be noted that, unless otherwise indicated, when it is said that (i) the amino acid sequence of the invention has a degree of sequence identity with SEQ ID NO: 1 or 2 of at least 85%, preferably at least 90%, more preferably at least 95% (where CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, required by a particular aspect, are not taken into account when determining the degree of identity sequences) ; and/or when it is said that (ii) the amino acid sequence of the invention has no more than 7, preferably no more than 5, e.g. only 3, 2, or 1 amino acid differences with SEQ ID NO: 1 or 2 (again, excluding any C-terminal extension that may be present, and not including mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect), this also includes the sequences , which have no amino acid differences with the sequence of SEQ ID NO: 1 or 2, except for mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 (as required by a particular aspect ) and any C-terminal extensions that may be present.

Thus, in one specific aspect of the invention, PD1 binding molecules of the invention comprising the amino acid sequence of SEQ ID NO: 1 or 2, but with at least one amino acid mutation at positions 1, 11, 14, 52a, 73, 74, 83, 89 , 100a, 110 and/or 112 may have 100% sequence identity with SEQ ID NO: 1 or 2 (including their CDR1, CDR2 and CDR3, optionally containing the W52aV and/or W100aF mutation, but not including the mutations) or combinations of mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 disclosed herein and/or any C-terminal extension that may be present) and /or may have no amino acid difference from SEQ ID NO: 1 or 2 (i.e. other than a mutation(s) or combination of mutations at positions 1, 11, 14, 52a, 73, 74, 83, 83, 89, 100a, 110 and /or 112 disclosed herein and any C-terminal extension that may be present).

- 21 041987

When any amino acid differences are present (e.g. other than any C-terminal extension and mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 as required by a particular aspect of the present invention) , these amino acid differences may be present in the CDRs and/or in the framework regions, but they are preferably only present in the framework regions (defined by the Abm system, i.e. not in the CDRs defined by the Abm system), i.e. they are such that PD1 binding molecules of the invention have the same CDRs (determined by the Abm system) as those present in SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104, or 105.

In addition, when the PD1 binding molecule of the invention according to any aspect of the invention has one or more amino acid differences from the sequence of SEQ ID NO: 1 or 2 (other than mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89 , 100a, 110 and/or 112 as required by a particular aspect), then some specific but non-limiting examples of such mutations/amino acid differences that may be present (i.e. compared to the sequences of SEQ ID NO: 1 or 2) are, for example, humanizing substitutions; for example, see WO 09/138519 (or the prior art referred to in WO 09/138519) and WO 08/020079 (or the prior art referred to in WO 08/020079), as well as tables A-3 - A- 8 of WO 08/020079 (which are lists showing possible humanizing substitutions).

In addition, when the PD-1 binding fragments of the invention are N-terminal and/or form the N-terminal portion of the polypeptide in which they are present, then it is preferred that they contain a D at position 1 (i.e., an E1D mutation compared to the reference sequence A). A preferred, but non-limiting example of such an N-terminal PD-1 binding element is SEQ ID NO: 24 or 57. Accordingly, in the following aspect, the invention provides a polypeptide (described hereinafter) that has a PD-1 binding moiety (which is further described herein) at its N-terminus, wherein said PD-1 binding fragment has a D at position 1 and is, for example, SEQ ID NOs: 24, 25, 57, or 101-105.

Similarly, when the PD-1 binding molecule of the invention is used in a monovalent format, it is preferred that it has both the C-terminal extension X(n) described herein and the D at position 1. A preferred but non-limiting example of such a monovalent PD-1 binding molecule is shown as SEQ ID NO: 40. Accordingly, in the following aspect, the invention provides a univalent PD-1 binding molecule of the invention (described hereinafter) that has a D at position 1 and a C-terminal extension X ( n) (which is preferably one Ala residue). In one specific aspect, said monovalent PD-1 binding molecule is SEQ ID NO: 40, 101, or 102.

As preferred, but non-limiting examples, SEQ ID NOs: 23, 24, 39, 40, and 57 are examples of PD-1 binding molecules of the invention having amino acid differences from SEQ ID NO: 1 or 2, such as A14P, A74S, and /or K83R (in addition, as indicated in the previous paragraphs, SEQ ID NO: 24 and 40 and 57 also have an E1D mutation). Thus, in a particular aspect, the invention relates to PD-1 binding molecules of the invention (namely, having the mutations at positions 11, 89, 110 and/or 112 described herein, and, in addition, also being as described herein) that have at least a suitable combination of an optional E1D mutation, an A14P mutation, an A74S mutation, a K83R mutation, and/or an I89L mutation, and preferably a suitable combination of any two of these mutations, e.g., all of these mutations.

The PD1 binding molecules of the invention when used in a monovalent format and/or when they are at the C-terminus and/or form the C-terminus of the polypeptide in which they are present (or when they conversely have an open C-terminus in such a polypeptide, which generally means that the C-terminus of the ISVD is not associated or linked to a constant domain (such as the CH1 domain)); (see WO 12/175741 and PCT/EP2015/060643 (WO-015/173325)), preferably also have a C-terminal extension (X)n, where n is from 1 to 10, preferably from 1 to 5, eg 1, 2, 3, 4 or 5 (and preferably 1 or 2, eg 1); and each X is an amino acid residue (preferably naturally occurring) independently selected from naturally occurring amino acid residues (although in one preferred aspect it does not contain any cysteine residues) and preferably independently selected from the group consisting of alanine ( A), glycine (G), valine (V), leucine (L) or isoleucine (I).

According to some preferred but non-limiting examples of such C-terminal extensions X(n), X and n may be as follows:

(a) n=1 and X=Ala;

(b) n=2 and each X=Ala;

(c) n=3 and each X=Ala;

(d) n=2 and at least one X=Ala (with the remaining amino acid residues X independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

(e) n=3 and at least one X=Ala (with the remaining amino acid residues X independently selected from any natural amino acid, but preferably independently selected from Val,

- 22 041987

Leu and/or Ile);

(f) n=3, and at least two X=Ala (with the remaining X amino acid residues independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

(g) n=1 and X=Gly;

(h) n=2 and each X=Gly;

(i) n=3 and each X=Gly;

(j) n=2 and at least one X=Gly (with the remaining X amino acid residues independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

(k) n=3, and at least one X=Gly (with the remaining X amino acid residues independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

(l) n=3, and at least two X=Gly (with the remaining X amino acid residues independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

(m) n=2 and each X=Ala or Gly;

(n) n=3 and each X=Ala or Gly;

(o) n=3 and at least one X=Ala or Gly (with the remaining amino acid residues X independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile); or (p) n=3 and at least two X=Ala or Gly (with the remaining amino acid residues X independently selected from any natural amino acid, but preferably independently selected from Val, Leu and/or Ile);

Moreover, aspects (a), (b), (c), (g), (h), (i), (m) and (n) are especially preferred, where aspects in which n=1 or 2 are preferred. , and aspects in which n=1 are particularly preferred.

It should also be noted that it is preferred that any C-terminal extension present in the PD1 binding molecule of the invention does not contain a (free) cysteine residue (unless said cysteine residue is used or intended for further functionalization, e.g. for PEGylation).

Some specific, but non-limiting examples of suitable C-end extensions are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA, or GAG.

When the PD1 binding molecules of the invention contain mutations at positions 110 or 112 (optionally in combination with mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89 and/or 100a described herein), C- terminal amino acid residues of framework region 4 (starting at position 109) may in an embodiment of the invention be as indicated in SEQ ID NOs: 1, 2, 9-40, 57, 100, 101, 103, 104, 105, 106, or 107 , but where the 5 C-terminal residues can be substituted as follows:

(i) if there is no C-terminal extension: VTVKS (SEQ ID NO: 42), VTVQS (SEQ ID NO: 43), VKVSS (SEQ ID NO: 44) or VQVSS (SEQ ID NO: 45); or (ii) if there is a C-terminal extension: VTVKSX( _n ) (SEQ ID NO: 46), VTVQSX( _n ) (SEQ ID NO: 47), VKVSSX(n) (SEQ ID NO: 48) or VQVSSX _W ( SEQ ID NO: 49), such as VTVKSA (SEQ ID NO: 50), VTVQSA (SEQ ID NO: 51), VKVSSA (SEQ ID NO: 52) or VQVSSA (SEQ ID NO: 53).

When the PD1 binding molecules of the invention contain or alternatively do not contain mutations at positions 110 or 112 (but only mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89 and/or 100a described herein document), the C-terminal amino acid residues of framework region 4 (starting at position 109) may in an embodiment of the invention be as indicated in SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 but where the 5 C-terminal residues can be substituted as follows:

(i) when there is no C-terminal extension: VTVSS (SEQ ID NO: 54) (as in SEQ ID NO: 1 or 2); or (ii) when there is a C-terminal extension: VTVSSX( _n ) (SEQ ID NO: 55), eg VTVSSA (SEQ ID NO: 56). In these C-terminal sequences, X and n are as specified herein for C-terminal extension.

Some preferred, but non-limiting examples of PD1 binding molecules of the invention are shown in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104, or 105, and each of these sequences forms another aspect of the invention. Of these, the PD1-binding molecules of SEQ ID NOs: 924, 57, 98, 99, 103, 104, and 105 do not have a C-terminal extension, and the PD1-binding molecules of SEQ ID NOs: 25-40, 101, and 102 contain a C-terminal alanine ( which is a preferred but non-limiting example of the C-terminal extension described herein).

Examples of PD1 binding molecules of the present invention include the amino acid sequence shown in SEQ ID NOs: 23, 24, 39, 40, 57, 98, 99, 101, 102, 103, 104, or 105.

Thus, in an embodiment of the invention, a PD1 binding molecule (e.g., an ISVD, such as a nanobody) contains:

CDR1 (according to Kabat), which is the amino acid sequence of IHAMG (SEQ ID NO: 3); And

CDR2 (according to Kabat), which is the amino acid sequence

VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V); And

CDR3 (according to Kabat), which is the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F);

and also has:

a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which their CDRs are optionally containing the W52aV and/or W100aF mutation, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect , are not taken into account when determining the degree of sequence identity);

and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined in the present description, and not taking into account any of the mutations described in the present description at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (wherein said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally has:

C-terminal extension (X) _n where n is 1 to 10, preferably 1 to 5, eg 1, 2, 3, 4 or 5 (and preferably 1 or 2, eg 1); and each X is an amino acid residue (preferably natural) that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine ( I);

where in an embodiment of the invention:

the amino acid residue at position 1 is E or D;

the amino acid residue at position 11 is L or V;

the amino acid residue at position 14 is A or P;

the amino acid residue at position 52a is W or V;

the amino acid residue at position 73 is N, S, P or Q;

the amino acid residue at position 74 is A or S;

the amino acid residue at position 83 is K or R;

the amino acid residue at position 89 is T, V, I, or L;

the amino acid residue at position 100a is W or F;

the amino acid residue at position 110 is T, K or Q; and the amino acid residue at position 112 is S, K or Q;

for example, where the PD1 binding molecule contains one or more of the following mutations:

(i) position 1 is E or D;

(ii) position 11 is V;

(iii) position 14 is P;

(iv) position 52a is V;

(v) position 73 is S, P or Q;

(vi) position 74 is S;

(vii) position 83 is R;

(viii) position 89 is L;

(ix) position 100a is F;

for example, contains the following set of mutations:

a) position 1 is D or E, position 11 is V, position 14 is P, position 74 is S, position 83 is R, and position 89 is L;

b) position 1 is D or E, position 11 is V, position 14 is P, position 52a is V; position 73 is S, P or Q, position 74 is S, position 83 is R, position 89 is L, and position 100a is F;

c) position 1 is D or E, position 11 is V, position 14 is P, position 74 is S, position 83 is R, and position 89 is L;

- 24 041987

d) position 89 is L and position 11 is V;

e) position 89 is L and position 110 is K or Q;

f) position 89 is L and position 112 is K or Q;

g) position 89 is L and position 11 is V and position 110 is K or Q;

h) position 89 is L and position 11 is V and position 112 is K or Q;

i) 110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and 100aF and optionally 1D or 1E; or in combination with 11V, 14P, 74S, 83R, 89L and optionally 1D or 1E;

j) 112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and 100a F and optionally 1D or 1E; or in combination with 11V, 14P, 74S, 83R, 89L and optionally 1D or 1E;

k) position 11 is V and position 110 is K or Q; or

l) Position 11 is V and position 112 is K or Q.

In a further aspect, the invention provides a PD1 binding molecule (eg, ISVD, eg, nanobody) having:

CDR1 (according to Kabat), which is the amino acid sequence

IKAMG (SEQ ID NO: 3); And

CDR2 (according to Kabat), which is the amino acid sequence

VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V); And

CDR3 (according to Kabat), which is the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); and also has a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as their CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account in determining the degree sequence identity) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which CDRs, any C-terminal extension that may be present, and mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not taken into account when determining the degree of sequence identity); and/or no more than 7, for example no more than 5, preferably no more than 3, for example only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence SEQ ID NO: 1 or 2 ( wherein said amino acid differences, if present, may be present in the framework regions and/or CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally has

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

wherein the PD1 binding molecule (e.g., an ISVD such as a nanobody) contains, in one embodiment, one or more of the following amino acid residues (i.e., mutations compared to the amino acid sequence of SEQ ID NO: 1 or 2) at the indicated positions ( Kabat numbering):

1D or 1E;

11V;

14P;

52aV;

73Q, 73P or 73S;

74S;

83R;

89T or 89L; or

100aF;

for example, contains the following set of mutations:

1D or 1E in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and/or 100aF;

1D or 1E in combination with 11V, 14P, 74S, 83R and/or 89L;

89L in combination with 11V;

89L in combination with 110K or 110Q;

89L in combination with 112K or 112Q;

89L in combination with 11V, 14P, 74S, 83R and 1D, and 1E;

- 25 041987

110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and/or 100aF, and 1D or 1E;

112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and/or 100aF, and 1D or 1E;

110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and/or 1D or 1E;

112K or 112Q in combination with 11V, 14P, 74S, 83R, 89L and/or 1D or 1E;

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; or

11V in combination with 112K or 112Q.

As already stated, when a PD1 binding molecule (e.g., an ISVD such as a nanobody) of the invention is used in a monovalent format and/or present at the C-terminus of a polypeptide (as described herein), it is preferred that the PD1 binding molecule has a C-terminal extension. X(n), wherein the C-terminal extension may be as described herein for the PD1 binding molecules of the invention and/or as described in WO 12/175741 or PCT/EP2015/060643 (WO-015/173325).

Some preferred but non-limiting examples of PD1 binding molecules (e.g. ISVD such as nanobody) of the invention are shown in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 and 105, and each of these amino acid sequences individually form a further aspect of the invention.

As already indicated, the invention includes PD1 binding molecules containing the amino acid sequences of SEQ ID NO: 1 or 2, but in which position 89 is T; or in which position 1 is E or D, position 11 is V, position 14 is P, position 52a is V, position 73 is P, S or Q, position 74 is S, position 83 is R , position 89 is L and/or position 100a is F; or in which position 11 is V and position 89 is L (optionally in combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation and in particular in combination with a 110K or 110Q mutation). In an embodiment of the invention, amino acid sequences are provided wherein position 11 is V and position 89 is L, optionally with a 110K or 110Q mutation.

Thus, in one preferred aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having:

CDR1 (according to Kabat), which is the amino acid sequence

IKAMG (SEQ ID NO: 3); And

CDR2 (by Kabat), which is the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V); and CDR3 (by Kabat), which is the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); and also having: a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as their CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account in determining sequence identity) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which CDR, any C-terminal extension that may be present, and mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not taken into account when determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having:

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

where in an embodiment of the invention:

the amino acid at position 1 is E or D;

the amino acid at position 11 is L or V;

- 26 041987 the amino acid at position 14 is A or P;

the amino acid at position 52a is W or V;

the amino acid at position 73 is N, S, P, or Q;

the amino acid at position 74 is A or S;

the amino acid at position 8 3 is K or R;

the amino acid at position 89 is I, T or L;

the amino acid at position 100a is W or F;

the amino acid residue at position 110 is T, K or Q (and preferably T); and the amino acid residue at position 112 is S, K, or Q (and preferably S).

In another preferred aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having:

CDR1 (according to Kabat), which is the amino acid sequence

IHAMG (SEQ ID NO: 3); And

CDR2 (according to Kabat), which is the amino acid sequence

VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V); And

CDR3 (according to Kabat), which is the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); and having:

the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as their CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account when determining the degree of sequence identity) at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not considered in determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110, and/or 112, which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having:

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

in which one or more of the following is true:

the amino acid at position 1 is E or D;

the amino acid at position 11 is L or V;

the amino acid at position 14 is A or P;

the amino acid at position 52a is W or V;

the amino acid at position 73 is S, P, N, or Q;

the amino acid at position 74 is A or S;

the amino acid at position 83 is K or R;

the amino acid at position 89 is I, T or L;

the amino acid at position 100a is W or F;

the amino acid residue at position 110 is T, K or Q (and preferably T); or the amino acid residue at position 112 is S, K or Q (and preferably S).

In one specific, but non-limiting aspect, PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one or more of the following amino acid residues (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (Numbering according to Kabat):

11V in combination with 89L;

11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L and/or 100aF and optionally 1D;

11V in combination with 14P, 74S, 83R, 89L and 1D or 1E;

11V combined with 110K or 110Q;

11V combined with 112K or 112Q;

11V in combination with 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L, 100aF, 110K or 110Q, and/or 1D or 1E;

- 27 041987

11V in combination with 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L, 100aF, 112K or 112Q, and/or 1D or 1E;

11V in combination with 89L and 110K or 110Q;

11V in combination with 89L and 112K or 112Q; or

11V in combination with 1D or 1E, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and/or 100aF;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g. Kabat) and have a common degree sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one or more of the following amino acid residues (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (Numbering according to Kabat):

89L in combination with 11V;

89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 100aF and/or 1D or 1E;

89L in combination with 11V, 14P, 74S, 83R and/or 1D or 1E;

89L combined with 110K or 110Q;

89L combined with 112K or 112Q;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 110K or 110Q, and/or 1D or 1E;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 112K or 112Q, and/or 1D or 1E;

89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q, and/or 1D or 1E;

89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q, and/or 1D or 1E;

89L in combination with 11V and 110K or 110Q; or

89L in combination with 11V and 112K or 112Q;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g. Kabat) and have a common degree sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one or more of the following amino acid residues (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (Numbering according to Kabat):

110K or 110Q combined with 1D;

110K or 110Q combined with 1E;

110K or 110Q combined with 11V;

110K or 110Q in combination with 14P;

110K or 110Q combined with 52aV;

110K or 110Q in combination with 73S or 73Q, or 7 3P;

110K or 110Q combined with 74S;

110K or 110Q combined with 83R;

110K or 110Q combined with 89L;

110K or 110Q combined with 100aF;

110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L, 100aF and/or 1D or 1E;

110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and/or 1D or 1E;

110K or 110Q combined with 8 9L; or

110K or 110Q combined with 11V and 89L;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g. Kabat) and have a common degree sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one or more of the following amino acid residues (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (Numbering according to Kabat):

112K or 112Q combined with 1D or 1E;

112K or 112Q combined with 11V;

112K or 112Q in combination with 14P;

112K or 112Q combined with 52aV;

112K or 112Q in combination with 73S or 73Q or 73P;

- 28 041987

112K or 112Q combined with 74S;

112K or 112Q combined with 83R;

112K or 112Q combined with 89L;

112K or 112Q combined with 100aF;

112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L, 100aF and/or 1D or 1E;

112K or 112Q in combination with 11V, 14P, 74S, 83R, 89L and/or 1D or 1E; or

112K or 112Q combined with 11V and 89L;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g. Kabat) and have a common degree sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain a T at position 89 and have CDRs that are the same as those present in the sequence of SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104, or 105 (eg, Kabat) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain V at position 11 and L at position 8-9, and have CDRs that are the same as the CDRs that are present in the sequence of SEQ ID NO: 9 -40, 57, 100, 101, 102, 103, 104, or 105 (e.g. Kabat), and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

As already indicated, it is preferred that the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention according to the above aspects further comprise a suitable combination of an A14P mutation, an A74S mutation, and/or a K83R mutation, and preferably a suitable combination of any two these mutations, for example, all three of these mutations (for example, E1D (optional), L11V, A14P, W52aV, N73S or N73Q or N73P, A74S, K83R, I89L and W100aF). When a LAG3-binding or HSA-binding fragment is present at the N-terminus of the PD1-binding molecule of the present invention, it is preferred that the N-terminal binding fragment contains the E1D mutation.

In another aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having

CDR1 (by Abm), which is the amino acid sequence

GSIASIHAMG (SEQ ID NO: 6); And

CDR2 (by Abm), which is the amino acid sequence

VITXSGGITY (SEQ ID NO: 7, where X is W or V); And

CDR3 (by Abm), which is the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F);

and also having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account when determining the degree of identity sequences) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which the CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not considered in determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

where in an embodiment of the invention:

the amino acid at position 1 is E or D;

- 29 041987 the amino acid at position 11 is L or V;

the amino acid at position 14 is A or P;

the amino acid at position 52a is W or V;

the amino acid at position 73 is N, S, P, or Q;

the amino acid at position 74 is A or S;

the amino acid at position 83 is K or R;

the amino acid at position 89 is V, I, T, or L;

the amino acid at position 89 is T;

the amino acid at position 100a is W and F;

the amino acid residue at position 110 is T, K or Q (and preferably T); and the amino acid residue at position 112 is S, K or Q (and preferably S);

for example, in an embodiment, the PD1 binding molecule contains one or more of the following mutations: (i) position 1 is D or E; (ii) position 11 is V; (iii) position 14 is P; (iv) position 52a is V; (v) position 73 is Q, P or S; (vi) position 74 is S;

(vii) position 83 is R; (viii) position 89 is L or T; (ix) position 100a is F; for example, contains the following set of mutations:

a) position 11 is V; position 14 is P; position 52a represents V; position 73 is P, S or Q; position 74 is S; position 83 is R; position 89 is L; position 100a is F; and optionally position 1 is E or D;

b) position 11 is V; position 14 is P; position 74 is S; position 83 is R; position 89 is L; and optionally position 1 is E or D;

c) position 89 is L and position 11 is V;

d) position 89 is L and position 110 is K or Q;

e) position 89 is L and position 112 is K or Q;

f) position 11 is V; position 14 is P; position 52a represents V; position 73 is P, S or Q; position 74 is S; position 83 is R; position 89 is L; position 100a is F; position 110 is K or Q and optionally position 1 is E or D;

g) position 11 is V; position 14 is P; position 52a represents V; position 73 is S, P or Q; position 74 is S; position 83 is R; position 89 is L; position 100a is F; position 112 is K or Q and optionally position 1 is E or D;

h) position 11 is V; position 14 is P; position 74 is S; position 83 is R; position 89 is L; position 110 is K or Q and optionally position 1 is E or D;

i) position 11 is V; position 14 is P; position 74 is S; position 83 is R; position 89 is L; position 112 is K or Q and optionally position 1 is E or D;

j) position 89 is L and position 11 is V and position 110 is K or Q;

k) position 89 is L and position 11 is V and position 112 is K or Q;

l) position 11 is V and position 110 is K or Q; or

m) position 11 is V and position 112 is K or Q.

In a further aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having

CDR1 (by Abm), which is the amino acid sequence

GSIASIHAMG (SEQ ID NO: 6); And

CDR2 (by Abm), which is the amino acid sequence

VITXSGGITY (SEQ ID NO: 7, where X is W or V); And

CDR3 (by Abm), which is the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F);

and also having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account when determining the degree of identity sequences) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which the CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or

- 30 041987

2 required by a particular aspect are not taken into account when determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

wherein the immunoglobulin single variable domain contains one or more of the following amino acid residues (i.e., mutations compared to the amino acid sequence of SEQ ID NO: 1 or 2) at the indicated positions (Kabat numbering):

1D or 1E;

11V;

14P;

52aV;

73Q, 73S or 73P;

74S;

83R;

89L or 89T; or

100aF;

for example, contains the following set of mutations:

89T or 89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF or 1E, or 1D;

89T or 89L in combination with 11V, 14P, 74S, 83R or 1E or 1D;

89L in combination with 11V;

89L combined with 110K or 110Q;

89L in combination with 112K or 112Q;

89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q or 1E or 1D;

89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q, or 1E or 1D;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 110K or 110Q, or 1E or 1D;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 112K or 112Q, or 1E or 1D;

89L in combination with 11V and 110K or 110Q;

89L in combination with 11V and 112K or 112Q;

11V combined with 110K or 110Q; or

11V combined with 112K or 112Q.

As already stated, when a PD1 binding molecule (e.g., an ISVD such as a nanobody) of the invention is used in a monovalent format and/or when a PD1 binding moiety is present at the C-terminus of the polypeptide (as described herein), it is preferred that the PD1 binding fragment had a C-terminal extension X(n), wherein the C-terminal extension may be as described herein for the PD1 binding molecules of the invention and/or as described in WO 12/175741 or PCT/EP2015/060643 (WO-015/173325).

Some preferred but non-limiting examples of PD1 binding molecules (e.g., ISVD, such as a nanobody) of the invention are shown in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104, or 105, and each of these amino acid sequences individually form a further aspect of the invention.

As already indicated, in the invention the amino acid sequences in which position 89 is T; or in which position 1 is E or D, position 11 is V, position 14 is P, position 52a is V, position 73 is P, S or Q, position 74 is S, position 83 is R , position 89 is L and/or position 100a is F; or in which position 11 is V and position 89 is L (optionally in a suitable combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation and in particular in combination with a 110K or 110Q mutation) are part of this inventions. In an embodiment of the invention, the amino acid sequence at position 11 has a V, and at position 89 has an L, optionally with a 110K or 110Q mutation.

- 31 041987

Thus, in one preferred aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having:

CDR1 (by Abm), which is the amino acid sequence

GSIASIHAMG (SEQ ID NO: 6); And

CDR2 (by Abm), which is the amino acid sequence

VITXSGGITY (SEQ ID NO: 7, where X is W or V); And

CDR3 (by Abm), which is the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F);

and also having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account when determining the degree of identity sequences) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which the CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not considered in determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

in which, in an embodiment of the invention, the amino acid residue at position 1 is E or D;

the amino acid residue at position 11 is L or V;

the amino acid residue at position 14 is A or P;

the amino acid residue at position 52a is W or V;

the amino acid residue at position 73 is S, P, N, or Q;

the amino acid residue at position 74 is A or S;

the amino acid residue at position 83 is K or R;

the amino acid residue at position 89 is T, L or I;

the amino acid residue at position 100a is W or F;

the amino acid residue at position 110 is T, K or Q (and preferably T); and the amino acid residue at position 112 is S, K, or Q (and preferably S).

In yet another preferred aspect, the invention provides a PD1 binding molecule (e.g., an immunoglobulin single variable domain (ISVD), such as a nanobody) having

CDR1 (by Abm), which is the amino acid sequence

GSIASIHAMG (SEQ ID NO: 6); And

CDR2 (by Abm), which is the amino acid sequence

VITXSGGITY (SEQ ID NO: 7, where X is W or V); And

CDR3 (by Abm), which is the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F);

and also having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be present, as well as CDRs, optionally containing the W52aV and/or W100aF mutation, are not taken into account when determining the degree of identity sequences) of at least 85%, preferably at least 90%, more preferably at least 95% (e.g. 100%) (in which the CDR, any C-terminal extension that may be present, as well as mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by a particular aspect are not considered in determining the degree of sequence identity); and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not considering any of the mutations,

- 32 041987 data herein at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112, which may be present, and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but preferably only present in the framework regions and not in the CDRs);

and optionally having

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I );

in which, for example, one or more of the following is true:

the amino acid residue at position 1 is E or D;

the amino acid residue at position 11 is V;

the amino acid residue at position 14 is P;

the amino acid residue at position 52a is V;

the amino acid residue at position 73 is S, P or Q;

the amino acid residue at position 7 4 is S;

the amino acid residue at position 8 3 is R;

the amino acid residue at position 89 is L;

the amino acid residue at position 100a is F;

the amino acid residue at position 110 is T, K or Q; or the amino acid residue at position 112 is S, K or Q.

In one specific, but non-limiting aspect, PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one of the following sets of mutations (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (numbering according to Kabat): 11V in combination with 89L;

11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L, 100aF and 1E or 1D;

11V in combination with 14P, 74S, 83R, 89L and 1E or 1D;

11V combined with 110K or 110Q;

11V combined with 112K or 112Q;

11V in combination with 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L, 100aF, 110K or 110Q, and 1E or 1D;

11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R, 89L, 100aF, 112K or 112Q, and 1E or 1D;

11V in combination with 14P, 74S, 83R, 89L, 110K or 110Q, and 1E or 1D;

11V in combination with 14P, 74S, 83R, 89L, 112K or 112Q, and 1E or 1D;

11V in combination with 89L and 110K or 110Q; or

11V in combination with 89L and 112K or 112Q;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104, or 105 (for example, by Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one of the following sets of mutations (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (numbering according to Kabat): 89L in combination with 11V;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF and 1E or 1D;

89L in combination with 11V, 14P, 74S, 83R and 1E or 1D;

89L combined with 110K or 110Q;

89L combined with 112K or 112Q;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 110K or 110Q and 1E or 1D;

89L in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 100aF, 112K or 112Q and 1E or 1D; 89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q and 1E or 1D;

89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q and 1E or 1D;

89L in combination with 11V and 110K or 110Q; or

89L in combination with 11V and 112K or 112Q;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104, or 105 (for example, by Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one of the following sets of mutations (i.e., mutations compared to the sequence of SEQ ID NO: 1 or 2) at the indicated positions (numbering according to Kabat):

- 33 041987

110K or 110Q combined with 11V;

110K or 110Q combined with 89L;

110K or 110Q combined with 11V and 89L;

110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and 100aF, and 1E or 1D;

110K or 110Q combined with 11V, 14P, 74S, 83R, 89L and 1D; or

110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and 1E, and have CDRs that are the same as those present in the sequence of SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (eg, Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another specific, but non-limiting aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain one or more of the following sets of mutations (i.e., mutations compared to the sequence of SEQ ID N0: 1 or 2) at the indicated positions (Numbering according to Kabat):

112K or 112Q combined with 11V;

112K or 112Q combined with 89L;

112K or 112Q combined with 11V and 89L;

112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q, or 73P, 74S, 83R, 89L and 100aF, and IE or 1D; or

112K or 112Q combined with 11V, 14P, 74S, 83R, 89L and 1E or 1D;

and have CDRs that are the same as the CDRs that are present in SEQ ID NOs: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104, or 105 (for example, by Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain a T or L at position 89 and have CDRs that are the same as those present in the sequence of SEQ ID NOs: 1, 2, 9- 40, 57, 98, 99, 101, 102, 103, 104, or 105 (eg, Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein.

In another aspect, the PD1 binding molecules (e.g., ISVDs such as nanobodies) of the invention contain V at position 11 and L at position 89, and have CDRs that are the same as those present in the sequence of SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104, or 105 (e.g., Abm) and share a common degree of sequence identity with the amino acid sequences of SEQ ID NO: 1 or 2 as described herein .

As already indicated, it is preferred that the PD1 binding molecules of the invention according to the above aspects further comprise a suitable combination of E1D mutation, L11V mutation, A14 mutation, W52aV mutation, N73S mutation, or N73Q mutation, or N73P mutation, A7 4S mutation, mutation K83R, I89L mutations and/or W100aF mutations, and preferably a suitable combination of any two of these mutations, eg all of these mutations. When the PD1 binding molecule is monovalent, or its PD1 binding fragment is present at the N-terminus of the polypeptide, it is preferred that it also has an E1D mutation.

In another specific, but non-limiting aspect, the invention relates to a single immunoglobulin variable domain that contains an amino acid sequence or consists essentially of an amino acid sequence selected from one of the following amino acid sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 SEQ

ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:

26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ

ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, SEQ ID

NO: 39, SEQ ID NO: 40, SEQ ID NO: 57, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.

In another specific, but non-limiting aspect, the invention relates to a single immunoglobulin variable domain that is an amino acid sequence or consists essentially of an amino acid sequence selected from one of the following amino acid sequences: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 57, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO : 104 and SEQ ID NO: 105.

Also, as already indicated herein, the amino acid residues of a PD1 binding molecule (eg, ISVD, such as a nanobody) are numbered according to the general numbering for VH proposed in Rabat et al. (Sequence of proteins of immunological interest, US Public Health Services, NIH Bethesda, MD, Publication No. 91) and applied to camelid VHH domains in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or referenced in this document. It should be noted that, as is well known in the art for VH- and for VHH

- 34 041987 domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues specified in the Kabat numbering system (i.e. one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This means that, in general, Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the real sequence. However, in general, according to Kabat numbering and regardless of the number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the beginning of FR1 and, reciprocally, position 36 according to Kabat numbering corresponds to the beginning of FR2 and, reciprocally, position 66 according to Kabat numbering corresponds to the beginning of FR3 and, conversely, position 103, according to Kabat numbering, corresponds to the beginning of FR4.

Alternative methods for numbering amino acid residues of VH domains, which can also be applied similarly to camelid and nanobody VHH domains, is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called AbM definition and the so-called contact definition. However, in the present specification, aspects and figures, the Kabat numbering adapted to the VHH domains by Richman and Muilderman will be followed unless otherwise indicated.

These and other aspects, embodiments, advantages, uses and uses of the invention will be apparent from the further description herein.

Accordingly, in a further aspect, the invention relates to polypeptides or other chemical entities that contain or essentially consist of at least one (such as one, two or three) PD1 binding fragment described herein.

PD1 binding molecules (eg, ISVDs such as nanobodies) of the invention may be fused to one or more other amino acid sequences, entities, or fragments. These other amino acid sequences, chemical entities, or fragments may confer one or more desired properties on the resulting PD1 binding molecules of the invention and/or may alter the desired properties of the resulting PD1 binding molecules of the invention, for example, providing the resulting PD1 binding molecules of the invention with the desired biological and/or therapeutic activity (e.g., providing the resulting PD1-binding molecules of the invention with affinity and preferential efficacy against another therapeutically important target such that the resulting polypeptide becomes bispecific for PD1 and another therapeutically relevant target such as CTLA4, LAG3, BTLA or CD27), providing the desired half-life and/or otherwise modifying or improving pharmacokinetic and/or pharmacodynamic properties by targeting the PD1 binding molecule to specific cells, tissues, or organs (including cancer cells and cancer tissues) to provide a cytotoxic effect and/or to act as a detectable tag or label. Some non-limiting examples of such other amino acid sequences, chemical entities or fragments are given below:

one or more suitable linkers (such as a 9GS, 15GS or 35GS linker (any combination of 9, 15, 20 or 35 amino acids G and S, such as, for example,

GGGGGGGS (9GS linker, SEQ ID NO: 125),

GGGGGGGGGGGGGGGGGGS (20GS linker, SEQ ID NO: 100) or

GGGGGGGGGGGGGGGGGGGGS (35GS linker, SEQ ID NO: 58)) or (GGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); and/or one or more binding fragments, binding domains, or binding elements that are directed against a therapeutically relevant target other than PD1 (i.e., to provide a PD1 binding molecule (e.g., an ISVD, such as a nanobody) of the invention that is bispecific in against PD1 and another therapeutically significant target, for example, against another epitope of PD1, CD27, LAG3, CTLA4, BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1, PD-L2, ILT1, ILT2 SEACAM1, SEACAM5, TIM3, TIGIT, VISTA, ILT3, ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40, CD137, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, GNK2C2A, , NKG2E, IL-10, IL-17, TSLP); and/or one or more binding domains or binding elements that provide an increase in half-life (for example, a binding domain or binding element that can bind to a serum protein, such as serum albumin, for example, human serum albumin), such as ALB11002; and/or one or more binding domains or binding elements that direct the PD1 binding molecule (eg, an ISVD, such as a nanobody) to a desired cell, tissue, or organ (eg, a cancer cell); and/or one or more binding domains or binding elements that provide increased specificity for PD1 (they can typically bind to PD1, but generally cannot be functional against PD1 on their own); and/or a binding domain that binds an element or other chemical entity that allows

- 35 041987 a PD1 binding molecule (eg, ISVD, such as a nanobody) to be internalized into the desired cell (eg, an anti-EGFR internalizing nanobody described in WO 05/044858); and/or a moiety that increases the half-life, such as a suitable polyethylene glycol group (i.e., PEGylation) or an amino acid sequence that increases the half-life, such as human serum albumin or a suitable fragment (e.g., a fusion with albumin) or, for example , the serum albumin binding peptide described in WO 2008/068280; and/or a payload such as a cytotoxic payload; and/or a detectable label or tag, such as a radioactive label or a fluorescent label; and/or TEG, which can contribute to immobilization, detection and/or cleaning of the binding PD1 molecules (for example, ISVD, such as nanothel), such as HIS (for example, NNNNNNNEN, SEQ ID NO: 93, or NNNNNNNNNNNNNENNNNENNE; SeQ ID no: 94 ) or FLAG (DYKDDDK (SEQ ID NO: 95)) (for example, FLAG3); and/or a tag that can be functionalized, such as a C-terminal GGC or GGGC tag; and/or

C-terminal extension X(n) (e.g. -Ala), which may be an extension further described herein for PD1 binding molecules (e.g. ISVD such as nanobodies) of the invention and/or described in WO 12/175741 or PCT/EP2015/060643 (WO-015/173325).

Included within the scope of the invention is a PD1 binding molecule (e.g., an ISVD, such as a nanobody) that includes one or more antibody (preferably human) moieties or fragments, e.g., an Fc portion or a functional fragment thereof, or one or more constant domains, and/ or from camelid antibodies consisting only of the heavy chain (eg, one or more constant domains).

LAG3 binding molecules.

The present invention relates to improved LAG3 binding molecules, eg improved anti-LAG3 ISVDs and in particular improved LAG3 nanobodies, eg 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L); F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L); F0237611B09 (E1D, L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L)-35GS-ALB11002-A; or F0237611B09 (E1D, L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L)-35GS-F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L)-35GS-ALB11002.

As discussed, the LAG3 binding molecules of the present invention are any of the molecules described herein that bind to LAG3 (e.g., ISVD, such as nanobodies), as well as any polyvalent or polyspecific binding molecule (e.g., PD1/LAG3 binding), which includes such a molecule fused to another binding molecule. For example, a LAG3 binding molecule may include a LAG3 binding moiety fused to a moiety that binds to PD1, CD27, CTLA4, HSA, BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1, PD- L2, ILT1, ILT2 CEACAM1, CEACAM5, TIM3, TIGIT, VISTA, ILT3, ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40, CD137, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3 , NKG2A, NKG2C, NKG2E, IL-10, IL-17, TSLP. An individual LAG3 binding moiety may be referred to as a LAG3 binding moiety if it is part of a larger molecule, such as a polyvalent molecule, where the LAG3 binding moiety is fused to another binding moiety, such as F023700924 or F023700931.

The LAG3 binding molecules of the present invention include polypeptides that are variants of polypeptides containing the amino acid sequence of SEQ ID NO: 63, but which are mutated at positions 1, 11, 14, 41, 43, 62, 74, 83 and/or 89.

As further described herein, the LAG3 binding molecules of the invention preferably have the same combination of CDRs (i.e., CDR1, CDR2, and CDR3) that are present in 11B09 or in a binding molecule containing the sequence 11B09 (SEQ ID NO : 63). See table. IN 1.

The present invention also includes LAG3 binding molecules that are 11B09 variants that contain the amino acid sequence shown in Table B-2 below. Included within the scope of the present invention are LAG3 binding molecules that include the CDR1, CDR2, and CDR3 of the variants shown in Table 1 below. AT 2.

In addition, the present invention includes PD1/LAG3 binding molecules containing a LAG3 binding fragment that includes CDR1, CDR2 and CDR3, or the 11B09 amino acid sequence or one of its variants shown in Table 1 below. AT 2.

- 36 041987

Table B-1

LAG3 binding molecule, 11B09

Description Subsequence air defense 9 (maybe be named in this document "F0237611B09" or "611B09") SEQ ID NO: 63 EVQLVE S GGGLVQAGGS LRL S CAAS GRTFSDYVMGWFRQARGNERE F VAAISESGGRTHYADAVKGRFTISRDNAKNTLYLQMNSLKPEDTAVY YCATTLLWTSEYAPIKANDYDYWGOGTLVTVS S

Table B-2

LAG3 binding molecules that are sequence-optimized variants of 11B09

Description Subsequence 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) Name: F023700842 SEQ ID NO: 64 EVQLVE SGGGWQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS CDR1 (SEQ ID NO: 65) GRTFSDYVMG or DYVMG CDR2 (SEQ ID NO: 66) AISESGGRTH or AISE SGGRTHYADXVKG; where X is A or S (e.g. AISESGGRTHYADAVKG or AISESGG RTHYADSVKG)

- 37 041987

CDR3 (SEQ ID NO: 67) TLLWWTSEYAPIKANDYDY Monovalent SO (optimized for sequences) 611B09+ALB11002 Name: F023701128 Description: F0237611B09(EID,L11V,A14P, R4IP,N4ZK,A62S,A74S,K83R,V 89L)-35GS-ALB11002-A Target: hLAG-3 SEQ ID NO: 96 DVQLVE S GGGWQP GGS LRL S CAAS GRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFTISR DNSKNTLYLOMNSLRPEDTALYYCATTLLWWTSEYA PIKANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGGWQPGNS LRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISG SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPE DTALYYCTIGGSLSRSSQGTLVTVSSA Bivalent SO (optimized for sequences) 611B09+ALB11002 Name: F023700962 Description: F0237611B09 (EID,L11V,A14P,R41P, N4 3K, A 62S,A74S,K83R,V89L)-35GS- F0237611B09 (L11V,A14P,R41P,N43K,A62S, A74S,K83R,V89L)-35GS- ALB11002-A Target: hLAG-3 SEQ ID NO: 97 DVQLVE S GGGWQP GGS LRL S CAAS GRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFTISR DNSKNTLYLOMNSLRPEDTALYYCATTLLWWTSEYA PIKANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGGWQPGGS LRL S CAAS GRTFSDYVMGW FRQAP GKE RE EVAAISE SGGRTHYADSVKGRFTISRDNSKNTLYLOMNSLRPE DTALYYCATTLLWWTSEYAPIKANDYDYWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGWQPGNSLRLSCAASGFTFSSFGM SWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSS QGTLVTVSSA Name: F023700594 Description: F0237611B09- 35GS-F0237611B09-35GS- ALB11002 Target: hLAG-3 SEQ ID NO: 127 EVQLVE S GGGLVQAGGS LRL S CAAS GRTFSDYVMGW FRQARGNEREFVAAISESGGRTHYADAVKGRFTISR DNAKNTLYLOMNSLKPEDTAVYYCATTLLWWTSEYA PIKANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRL S CAAS GRTFSDYVMGW FRQARGNE RE EVAAISE SGGRTHYADAVKGRFTISRDNAKNTLYLQMNSLKPE DTAVYYCATTLLWWTSEYAPIKANDYDYWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGM SWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS

*CDRs of LAG3 binding molecules are underlined and/or in bold

The present invention includes embodiments in which the LAG3 binding moiety of the invention (e.g., in a PD1/LAG3 binding molecule) comprises one, two, or three CDRs of the LAG3 binding molecule described in Table B-1 or B-2 above (e.g., SEQ ID NO: 63, 64, 95, 96 or 97), where each region contains 0, 1, 2, 3, 4 or 5 amino acid substitutions, e.g. conservative substitutions, and/or has 100, 99, 98, 97, 96 or 95% sequence identity with the CDR sequences shown in Table 1. B-1 or B-2, wherein the LAG3 binding molecule of the invention containing such CDRs retains the ability to bind to LAG3. In an embodiment, the first amino acid of the LAG3 binding molecule of the present invention is E. In one embodiment, the first amino acid of the LAG3 binding molecule of the present invention is E or D.

The present invention includes a LAG3 binding molecule F023700656 (11B09 (E1D), SEQ ID NO: 63 (E1D)).

Residue numbers according to the Kabat system for some of the residues of the LAG3-binding molecules listed in Table 1. B-1 or B-2, shown in sequence below:

EVQLVE SGGGV11VQP14GG SLRLSCAASG RTFSDYVMGW FRQAP41GK43ERE FVAAISESGG rthyads ₆₂ vkg rftisrdns ₇₄ k ntlylqmnsl r ₈₃ pedtal ₈₉ yyc attllwwtse yapikandyd YWGQGTLVTV SS (SEQ ID NO: 64). Optionally, remainder 1 is D.

The present invention includes any LAG3-binding molecule comprising the amino acid sequence of SEQ ID NO: 63, 64, or 127 (or having a LAG3-binding fragment of SEQ ID NO: 96 or 97) (as well as LAG3-binding molecules having an E1D or D1E mutation), or an amino acid a sequence having 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity (i.e. when comparing full-length amino acid sequences), where the LAG3 linker the molecule retains the ability to bind to LAG3 and optionally includes an HSA binding molecule.

The present invention includes LAG3 binding molecules such as LAG3 ISVDs (e.g. nanobodies) having CDR1, CDR2 and CDR3 of a binding molecule comprising the amino acid sequence shown in SEQ ID NO: 63, 64, 96, 97 or 127 (or a variant thereof, described herein), for example, containing the following CDRs:

CDR1, which contains the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65); And

CDR2, which contains the amino acid sequence AISESGGRTH (SEQ ID NO: 66); And

CDR3 which contains the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67);

and optionally having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 63, 64, 96, 97, or 127 (in which any C-terminal extension that may be present, as well as CDRs, are not taken into account in determining the degree of sequence identity) at least 85%, preferably at least 90%, more preferably at least 95% , 62, 74, 76, 83, 89, 100 and/or 105 are not taken into account in determining the degree of sequence identity);

and/or no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 amino acid differences (as defined herein, and not taking into account any of the mutations listed herein at positions 1 , 11, 14, 41, 43, 62, 74, 76, 83, 89, 100, and/or 105 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 63 , 64, 96, 97, or 127 (wherein said amino acid differences, if present, may be in the framework regions and/or CDRs, but are preferably only present in the framework regions and not in the CDRs);

and optionally

The C-terminal extension discussed herein, for example (X) _n , in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2 , 1); and each X is an amino acid residue (preferably natural) which is independently selected and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I ).

The present invention also includes LAG3 binding molecules fused to one or more half-life increasing molecules, such as ISVD, which binds to human serum albumin, such as ALB11002.

Polyspecific binding molecules.

The present invention includes PD1 binding and LAG3 binding molecules that can be fused into a single polyvalent multispecific molecule that binds to PD1 and LAG3 (PD1/LAG3 binding molecule), and in an embodiment of the invention such binding molecules are associated with one or more half-life increasing molecules that increase the half-life of the binding molecules in the subject's body. In an embodiment, the half-life increasing molecule is an ISVD (eg, nanobody) that specifically binds to human serum albumin (HSA), eg, ALB11002. In an embodiment, the polyspecific binding molecule is F023700899, F023700931, F023701016, F023701017, F023700924, F023700969,

- 39 041987

F023701163, F023701168, F023701173, F02370117, F023701176, 8, F023701161, F023701166, F023701171, F023701176, F023701162, F023701167, F023701172 или F023701177, описанные в настоящем документе.

In an embodiment, the PD1/LAG3 binding molecule comprises (1) a PD1 binding element comprising CDR1, CDR2, and CDR3 of any of the PD1 binding molecules listed in Table 1. A-1 or A-2, optionally each independently containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (e.g., conservative mutations), or

CDR1 containing the amino acid sequence

IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6);

CDR2 containing the amino acid sequence

VITXSGGITYYADSVKG (SEQ ID NO: 4, where X is W or V, e.g. W) or

VITXSGGITY (SEQ ID NO: 7, where X is W or V, eg W); And

CDR3 containing the amino acid sequence

DKHQSSXYDY (SEQ ID NO: 5, where X is W or F, eg W); and (2) a LAG3 binding element containing CDR1, CDR2 and CDR3 of any of the LAG3 binding molecules listed in Table. B-1 or B-2, optionally each independently containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (e.g., conservative mutations) or

CDR1 containing the amino acid sequence

GRTFSDYVMG (SEQ ID NO: 65);

CDR2 containing the amino acid sequence

AISESGGRTH (SEQ ID NO: 66); And

CDR3 containing the amino acid sequence

TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67), and optionally a half-life increasing fragment and/or a C-terminal extension, such as the HSA binding fragment specified herein, and/or optionally, a C-terminal extension, such as alanine.

In an embodiment, the PD1 binding fragment is as defined above under PD1 Binding Molecules, for example, containing the amino acid sequence of SEQ ID NO: 57, 98, 99, 103, 104, or 105 (optionally where residue 1 is E or D ). In an embodiment, the LAG3 binding element is as defined above under LAG3 Binding Molecules, for example, containing the amino acid sequence of SEQ ID NO: 63, 64, or 95 (optionally where residue 1 is E or D).

Multispecific binding molecules may include PD1 and LAG3 binding fragments and optionally an HSA binding element, as well as one or more binding fragments that bind to an additional antigen such as CD27, CTLA4, BTLA, TIM3, ICOS, B7-H3, B7- H4, CD137, GITR, PD-L1, PD-L2, ILT1, ILT2 , KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17 or TSLP.

When PD1 and/or LAG3 binding molecules (e.g., ISVDs such as nanobodies) contain one or more additional binding domains or binding elements (e.g., an additional substantially non-functional binding domain or element that binds to PD1 and/or LAG3, which provides increased specificity for PD1 and/or LAG3, binding domain or binding element for a therapeutic target other than PD1 and/or LAG3 (e.g. CTLA4, BTLA or CD27), binding domain or binding element for a target such as human serum albumin, that provides an extended half-life, and/or a binding domain or binding element that targets a PD1 and/or LAG3 binding molecule to a particular cell, tissue, or organ, and/or that allows the PD1 and/or LAG3 binding molecule to be internalized into the cell), these additional binding domains or binding elements preferably comprise one or more ISVDs (e.g. ep, nanobody) and, more preferably, all are ISVD. For example, and without limitation, these one or more additional binding domains or binding elements can be one or more nanobodies (including VHH, humanized VHH, and/or modified for camelid VH antibodies, such as modified for human camelid VH antibodies), single-domain antibody , which is a VH domain or a derivative of a VH domain, a dAb which is or essentially consists of a VH domain, or which is derived from a VH domain, or even a single domain antibody or dAb which is a VL domain or essentially consists of it. In particular, these one or more binding domains or binding elements, if present, may contain one or more nanobodies, and in particular, all are nanobodies. In an embodiment of the invention, an ISVD (eg, nanobody) that binds to a target other than PD1 and/or LAG3 binds to another target such as HSA, CTLA-4, BTLA, or CD27.

When the PD1 and/or LAG3 binding molecule (e.g., ISVD, such as nanobody) of the invention has an ISVD (e.g., nanobody) at its C-terminus (e.g., C-terminal ISVD binds to PD1

- 40 041987 and/or LAG3, or may, for example, be, if present in the polypeptide, an additional essentially non-functional ISVD against PD1 and/or LAG3, which provides increased specificity for PD1 and/or LAG3, ISVD to a therapeutic target, other than PD1 and/or LAG3, an ISVD to a target such as human serum albumin that gives an increase in half-life, or an ISVD that directs a PD1 and/or LAG3 binding molecule to a particular cell, tissue, or organ, and/or that allows binding PD1 and/or LAG3 molecule to be internalized into the cell), then the PD1 and/or LAG3 binding molecule (i.e., containing said C-terminal ISVD) preferably has a C-terminal extension X(n) (e.g., -Ala), wherein the C-terminal extension may be as described herein for the PD1 and/or LAG3 binding molecules of the invention and/or as described in WO 12/175741 or PCT/EP2015/060643 (WO2015/173325).

When the PD1 and/or LAG3 binding molecule contains, in addition to one or more moieties that bind to PD1 and/or LAG3, any other ISVDs (e.g. additional substantially non-functional ISVD to PD1 and/or LAG3, which provides increased specificity for PD1 and/or LAG3, ISVD to a therapeutic target other than PD1 and/or LAG3, ISVD to a target such as human serum albumin, which gives increased half-life, and/or an ISVD that targets a polypeptide of the invention to a particular cell, tissue, or organ, and/or that allows internalization of a polypeptide of the invention into a cell), and where such additional ISVDs are nanobodies or ISVDs that are VH sequences, or that essentially consist of and/or which are derived from VH sequences, then according to a preferred aspect of the invention, these one one or more such ISVDs (and preferably all) present in the PD1 and/or LAG3 binding molecule will contain in their sequence one or more framework region mutations that reduce binding to preexisting antibodies. In particular, according to this aspect of the invention, such additional ISVDs may contain a suitable combination of amino acid residues/mutations at positions 1, 11, 14, 74, 83, 89, 110 and/or 112, which are described in PCT/EP2015/060643 (WO2015/173325 ), and/or which are essentially as described herein for the PD1 and/or LAG3 binding molecules of the invention. In one specific aspect, when a PD1 and/or LAG3 binding molecule has such an ISVD at its C-terminus, then said ISVD that is present at and/or forms the C-terminus has framework mutations that reduce binding by pre-existing antibodies ; and said C-terminal ISVD will preferably also have a C-terminal extension X(n) (eg, -Ala) as described herein.

When the PD1 and/or LAG3 binding molecule is to have an increased half-life (i.e. compared to a monovalent binding molecule lacking a half-life increasing moiety, such as ALB11002), then the PD1 and/or LAG3 binding molecule preferably contains at least at least one (eg, one) ISVD (eg, nanobody) that provides such an extended half-life (eg, ALB11002). Such an ISVD in an embodiment of the invention is directed to a suitable serum protein such as transferrin or serum albumin (human). In particular, such an ISVD or nanobody can be a single-domain antibody or dAb to human serum albumin, as described, for example, in EP 2139918, WO 2011/006915, WO 2012/175400, WO 2014/111550. and may in particular be a serum albumin-binding nanobody as described in WO 2004/041865, WO 2006/122787, WO 2012/175400 or PCT/EP2015/060643 (WO2015/173325). Particularly preferred albumin-binding ISVDs are the Alb-1 nanobody (see WO 2006/122787) or humanized variants thereof such as Alb-8 (WO 2006/122787, SEQ ID NO: 62), Alb-23 (WO 2012/175400, SEQ ID NO: 1) and other humanized (and preferably also sequence optimized) variants of Alb-1 and/or variants of Alb-8 or Alb-23 (or, in general, ISVDs that have essentially the same CDRs, as Alb-1, Alb-8 and Alb-23).

As discussed, HSA binding molecules of the present invention are any molecules, such as those described herein, that bind to HSA (e.g., ISVDs such as nanobodies), as well as any polyvalent or polyspecific binding molecules that include such a molecule fused to another linking fragment. An individual HSA binding molecule may be referred to as an HSA binding moiety if it is part of a larger molecule, such as a polyvalent molecule, where the HSA binding moiety is fused to another binding moiety.

In an embodiment, the half-life increasing fragment is an ISVD (eg, nanobody) that binds to human serum albumin, eg, ALB11002, as described in Table 1 below. WITH.

As further described herein, the HSA binding molecules of the invention preferably have the same CDR combination (i.e., CDR1, CDR2, and CDR3) that is present.

- 41 041987 is in ALB11002, or contains the sequence of ALB11002 (SEQ ID NO: 59). See table. WITH.

The present invention also includes HSA-binding molecules that are variants of ALB11002 that contain the amino acid sequence shown in Table C below. The scope of the present invention includes HSA-binding molecules that include the CDR1, CDR2, and CDR3 of these variants shown in Table C below. WITH.

In addition, the present invention includes PD1/LAG3 binding molecules containing an HSA binding fragment that includes CDR1, CDR2 and CDR3, or the amino acid sequence of ALB11002, or one of its variants, shown below in table. WITH.

Table C

Nanobody ALB11002 to Human Serum Albumin (HSA)

SEQ ID NO Description Subsequence 59 ALBI1002 (may be referred to in this document as "ALB201") EVQLVESGGG XVQPGNSLRL SCAASGFTFS SFGMSWVRQA PGKGLEWVSS ISGSGSDTLY ADSVKGRFTI SRDNAKTTLY LQMNSLRPED TAXYYCTIGG SLSRSSOGTL VTVSSA; where X at positions 11 and 93 is L or V 60 CDR1 GFTFSSFGMS or SFGMS (amino acids 1-5 of SEQ ID NO: 60) 61 CDR2 SISGSGSDTLYADSVKG or SISGSGSDTL (amino acids 1-10 of SEQ ID NO: 61) 62 CDR3 GGSLSSR

Optionally, ALB11002 lacks the C-terminal alanine. Optionally, the HSA binding molecule contains the amino acid sequence shown in SEQ ID NO: 59, but which includes the mutation E1D, V11L and/or L93V, for example, contains the amino acid sequence

EVQLVESGGGWQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL

YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSS (SEQ ID NO: 59).

In an embodiment, the first amino acid of the HSA binding molecule of the present invention is E. In an embodiment, the first amino acid of the HSA binding molecule of the present invention is D.

The present invention includes embodiments in which one, two, or three CDRs of an HSA binding molecule each contains 0, 1, 2, 3, 4, or 5 amino acid substitutions, e.g., conservative substitutions and/or has 99, 98, 97, 96 or 95% sequence identity with the CDR sequences shown in Table C, where an HSA binding molecule having such CDRs retains the ability to bind to HSA.

In an embodiment, the half-life increasing fragment is an HSA-ISVD (eg, nanobody) containing:

CDR1 which contains the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60); And

CDR2 which contains the amino acid sequence SISGSGSDTL (SEQ ID NO: 61); And

CDR3 which contains the amino acid sequence GGSLSR (SEQ ID NO: 62);

and optionally having a degree of sequence identity with the amino acid sequence of SEQ ID NO: 59 (in which any C-terminal extension that may be present, as well as CDRs, are not taken into account in determining the degree of sequence identity) of at least 85%, preferably , at least 90%, more preferably at least 95% (wherein CDR, any C-terminal extension that may be present, is not taken into account in determining the degree of sequence identity); and/or no more than 7, e.g. no more than 5, preferably no more than 3, e.g. only 3, 2, or 1 amino acid differences (ignoring any C-terminal extension that may be present) with ami

- 42 041987 with the acid sequence of SEQ ID NO: 59 (wherein said amino acid differences, if present, may be present in the framework regions and/or in the CDRs, but are preferably only present in the framework regions and not in the CDRs);

and optionally having:

C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably natural) which is independently selected and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I) .

Such a human serum albumin-binding ISVD (eg, nanobody), when present, may contain in its sequence one or more framework region mutations that reduce binding by preexisting antibodies. In particular, when such human serum albumin-binding ISVD is a nanobody or single-domain antibody that essentially consists of and/or is derived from the VH domain, then the human serum albumin-binding ISVD may contain (suitable combination of) amino acid residues/mutations at positions 11 , 89, 110 and/or 112 which are described in PCT/EP2015/060643 (WO 02015/173325) and/or which are essentially as described herein for the PD1 binding molecules of the invention. For example, PCT/EP2015/060643 (WO-015/173325) describes a number of Alb-1, Alb-8 and Alb-23 variants that contain amino acid residues/mutations at positions 11, 89, 110 and/or 112 that reduce binding to pre-existing antibodies that can be used in the polypeptides of the invention.

When such a ISVD serum albumin-binding molecule (e.g., nanobody) is present at the C-terminus of the PD1 and/or LAG3 binding molecule, the ISVD serum albumin-binding molecule (and as a result the PD1 and/or LAG3-binding molecule of the invention) preferably has a C-terminal extension X ( n), wherein the C-terminal extension may be as described herein for the PD1 and/or LAG3 binding molecules of the invention and/or as described in WO 12/175741 or PCT/EP2015/060643 (WO2015/173325). For example, the C-terminal extension may be a single alanine residue. It also preferably has mutations that reduce binding to pre-existing antibodies, such as (suitable combination of) amino acid residues/mutations at positions 11, 89, 110 and/or 112 described in PCT/EP2015/060643 (WO2015/173325) .

However, as indicated, other means of increasing the half-life of a polypeptide of the invention (such as PEGylation, fusion with human albumin or a suitable fragment thereof, or use of a suitable albumin-binding peptide) are also within the scope of the invention.

In general, when a PD1 and/or LAG3 binding molecule of the invention has an extended half-life (e.g., due to the presence of a half-life-extending ISVD (e.g., nanobodies) or any other suitable method for increasing the half-life), the resulting PD1 and/or LAG3 binding the molecule of the invention preferably has a half-life (as defined herein) that is at least 2 times, preferably at least 5 times, such as at least 10 times or more than 20 times the period half-life of a PD1 and/or LAG3 binding molecule of the invention having no half-life increasing moiety (as measured in a human and/or in a suitable animal model such as mice or cynomolgus monkeys). In particular, the PD1 and/or LAG3 binding molecule of the invention preferably has a human half-life (as defined herein) of at least 1 day, preferably at least 3 days, more preferably at least 7 days, e.g. , at least 10 days.

It will be clear from this description that PD1-binding and LAG3-binding molecules that are based on one or more ISVDs (eg, nanobodies) may have different formats, ie. essentially be monovalent, divalent or trivalent, may be monospecific, bispecific, trispecific, and may be biparatopic (as defined herein and, for example, in WO 2009/68625). For example, a PD1 binding or LAG3 binding molecule of the invention may be: monovalent, ie containing a single PD1 or LAG3 binding moiety (eg, an ISVD, such as a nanobody). As already indicated, when used in a monovalent format, the PD1-binding or LAG3-binding molecule of the invention preferably has the C-terminal extension X(n) described above. Such a PD1 binding or LAG3 binding molecule of the invention may also have an extended half-life;

may be divalent or trivalent and monospecific. For example, in an embodiment of the invention, such a PD1-binding or LAG3-binding molecule of the invention comprises two or more ISVDs (eg, nanobodies) to PD1 or to LAG3, respectively, which may be the same or different; and when they are different, they may be directed against the same PD1 or LAG3 epitope, or against different epitopes on PD1 or LAG3 (in the latter case, to provide a biparatopic or polyparatopic PD1 binding or LAG3 binding molecule of the invention). Such a PD1 binding or LAG3 binding molecule of the invention

- 43 041987 may also have an extended half-life;

may be divalent, trivalent (or polyvalent) and bispecific or trispecific (or polyspecific). For example, in an embodiment, such a PD1 binding molecule of the invention will target PD1 and at least one other target such as, for example, CTLA-4, LAG-3, BTLA and/or CD27; for example, will contain a PD1 binding fragment and a CTLA4 binding fragment; a PD1 binding fragment and a BTLA binding fragment; a PD1 binding fragment and a LAG3 binding fragment (ie, will be a PD1/LAG3 binding molecule); or a PD1-binding fragment, a LAG3-binding fragment, and a BTLA-binding fragment. As described herein, said other target may be, for example, another therapeutically relevant target (i.e., other than PD1), thus providing a PD1 binding molecule of the invention that is bispecific for PD1 and the other therapeutic target. Said other target can also be a target that provides an extended half-life (such as human serum albumin), thus providing a PD1 binding molecule of the invention that has an extended half-life. As already indicated herein, such other target may also direct the PD1 binding molecule of the invention to specific cells, tissues, or organs, or may allow for the internalization of the PD1 binding molecule of the invention in the cell. It is also possible to combine these approaches/ISVD, for example, to provide a PD1 binding molecule of the invention that is bispecific for PD1 and at least one other therapeutically relevant target, and has an extended half-life.

It is further preferred that when these PD1-binding and LAG3-binding molecules comprise one or more ISVDs (e.g., nanobodies) other than the PD1-binding moiety or the LAG3-binding moiety, at least one, and preferably all of these other ISVDs, are contained in of its sequence, one or more frame region mutations that reduce binding to pre-existing antibodies (such as, in particular, a combination of amino acid residues/mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and /or 112, namely as described herein for PD1 binding and LAG3 binding molecules of the invention and/or as generally described in PCT/EP2015/060643 (WO-015/173325)). Also, when such PD1-binding or LAG3-binding molecules of the invention comprise at their C-terminus a PD1-binding moiety or a LAG3-binding moiety, respectively, then said C-terminal PD1-binding moiety or LAG3-binding moiety (and, as a result, the PD1-binding molecule or LAG3-binding moiety molecule of the invention) will preferably have the C-terminal extension X(n) described herein. Similarly, when such PD1-binding molecules and LAG3-binding molecules of the invention have a different ISVD at their C-terminus (i.e., not a PD1-binding fragment or a LAG3-binding fragment, but, for example, an ISVD that increases the half-life), then the indicated C-terminal ISVD ( and as a result, the PD1 binding molecule or LAG3 binding molecule of the invention will preferably have the C-terminal extension X(n) described herein and/or will contain in its sequence one or more frame region mutations that reduce binding to pre-existing antibodies (again, as further described herein and in PCT/EP 2015/060643 (WO 2015/173325)).

As will be appreciated by one of skill in the art, when a PD1 binding or LAG3 binding molecule (e.g., an ISVD, such as a nanobody) of the invention is intended for topical application (e.g., on the skin or eyes) or, for example, is to have a local therapeutic effect in somewhere, such as in the gastrointestinal tract (i.e. after oral or suppository administration) or in the lungs (i.e. after inhalation), or otherwise must be directly applied to its intended site of action ( eg, by direct injection), the PD1 binding molecule or LAG3 binding molecule of the invention does not generally require an increase in half-life. In addition, for the treatment of certain acute conditions or diseases, it may be preferable not to have a long half-life. In these cases, it will be preferable to use a monovalent PD1 binding molecule or LAG3 binding molecule according to the invention or another PD1 binding molecule or LAG3 binding molecule according to the invention (containing a PD1 binding or LAG3 binding element) without increasing the half-life, for example, a PD1 binding molecule or a LAG3 binding molecule according to the invention, which is bivalent or biparatopic with respect to PD1 or LAG3.

Some preferred, but non-limiting examples of such PD1 binding molecules and LAG3 binding molecules of the invention are schematically presented in Tables D-1a and D-1b below, and each is an additional aspect of the invention. Other examples of suitable polypeptides of the invention without increasing half-life will be apparent to the skilled artisan based on the description herein.

- 44 041987

Table D-1a

Schematic representation of some PD1 and/or LAG3 binding molecules of the invention without ISVD increasing half-life

[PD-1 binding fragment according to the invention] [Contact PD-1 fragment according to the invention]-X(p) [Linking snippet by . PD-1 Fragment of the Invention]-[Binding PD-1 of the Invention]-[Another Fragment]-[Another Fragment] [Linking snippet by . PD-1 Fragment of the Invention]-[Binding PD-1 of the Invention]-[Another Fragment]-[Another Fragment] [Linking fragment by X(n) PD-1 Fragment of the Invention]- [Binding PD-1 of the Invention]- [Another Fragment]- [Another Fragment]- [ Connecting fragment by X(n) PD-1 Fragment of the Invention]- [Binding PD-1 of the Invention]- [Another Fragment]- [Another Fragment]- [ Linking snippet by . PD-1 fragment of the invention]-X(p) according to the invention]- [Binding PD-1 [ Binder PD-1 fragment ps ) invention]-[Another snippet] [ Binder PD-1 fragment ι that invention]-[Another snippet]-

- 45 041987

X (η)

[Another Fragment]-[PD-1 Binding Fragment of the Invention]

[Other Fragment]-[PD-1 Binding Fragment of the Invention] X (n)

[PD-1 binding fragment according to the invention]-[Targeting unit]

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]

[PD-1 binding fragment according to the invention]-[Guiding fragment]-X (p)

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]-X(p)

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[Guiding Fragment]

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[Targeting Fragment]-X(p)

[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]

[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-X (p)

[PD-1 binding fragment according to the invention]

[PD-1 binding fragment of the invention]-X(n)

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]

[PD-1 binding fragment of the invention]-[PD-1 binding fragment of the invention]-X(n)

[PD-1 binding fragment according to the invention]-[Another fragment]

[PD-1 binding fragment according to the invention]-[Another fragment] X(n)

[Another Fragment]-[PD-1 Binding Fragment of the Invention]

[Other Fragment]-[PD-1 Binding Fragment of the Invention] X(n)

[PD-1 Binding Fragment of the Invention] - [Guide Fragment]

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]

- 46 041987

[PD-1 binding fragment according to the invention]-[Guiding fragment]-X (p)

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]-X(p)

Legend:

- "[PD-1 binding fragment of the invention]" represents a PD1 binding domain or fragment or element, such as ISVD 102C12 (EID, L11V, A14P, A74S, K83R, I89L) described herein

- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker

- “X(n)>> represents a C-terminal extension as described herein, such as a single alanine residue.

- "[Other Fragment]" represents a binding domain or binding element (eg, ISVD, such as a nanobody) binding an epitope that is different from PD1, for example, one or more of CTLA4-, BTLA-, LAG3-, and/or CD27- ISVD such as LAG3-ISVD, 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) described herein

- "[Targeting Fragment]" represents a binding domain or binding element (and in particular an ISVD such as a nanobody) that directs a polypeptide of the invention to a particular cell, tissue, or organ

D-lb table

Schematic representation of some PD1 and/or LAG3 binding molecules of the invention without ISVD increasing half-life

[LAG3 binding fragment according to the invention]

[LAG3 binding fragment according to the invention]-X(p)

[LAG3-binding fragment of the invention]-[LAG3-binding fragment of the invention]-[Another fragment]-[Another fragment] [LAG3-binding fragment of the invention]-[LAG3-binding fragment of the invention]-[Another fragment]-[Another fragment] [LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Another fragment]-[Another fragment]X(n)

- 47 041987

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Another fragment]-[Another fragment]X(n)

[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-X(n)

[LAG3 binding fragment according to the invention]-[Another fragment] [LAG3 binding fragment according to the invention]-[Another fragment]X(n)

[Another Fragment]-[LAG3 Binding Fragment of the Invention]

[Other Fragment]-[LAG3 Binding Fragment of the Invention] X(n)

[LAG3 binding fragment according to the invention]-[Guide fragment]

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]

[LAG3 binding fragment according to the invention]-[Guiding fragment]-X (n)

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]-X (p)

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Guiding fragment]

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Targeting fragment]-X(p)

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]-[LAG3 Binding Fragment of the Invention]

[Targeting Fragment]-[LAG3 Binding Fragment of the Invention]-[LAG3 Binding Fragment of the Invention]-X(n)

[LAG3 binding fragment according to the invention]

[LAG3 binding fragment according to the invention]-X(n)

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]

[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-X(n)

[LAG3 binding fragment according to the invention]-[Another fragment] [LAG3 binding fragment according to the invention]-[Another fragment]- 48 041987

X(η)

[Another Fragment]-[LAG3 Binding Fragment of the Invention]

[Other Fragment]-[LAG3 Binding Fragment of the Invention] X (n)

[LAG3 binding fragment according to the invention]-[Guide fragment]

[Targeting Fragment]-[LAG3 Binding Fragment of the Invention] ([LAG3 Binding Fragment of the Invention]-[Targeting Fragment] -X(n)

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]-X (p)

Legend:

- "[LAG3 binding fragment of the invention]" represents a LAG3 binding domain or fragment or element such as ISVD 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) described herein

- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker

- “X(n)>> represents a C-terminal extension as described herein, such as a single alanine residue.

- "[Other Fragment]" represents a binding domain or binding element (eg, ISVD, such as a nanobody) binding an epitope that is different from PD1, for example, one or more of CTLA4-, BTLA-, LAG3-, and/or CD27- ISVD such as PD1-ISVD 102C12 (EID, L11V, A14P, A74S, K83R, I89L) described herein

- "[Targeting Fragment]" represents a binding domain or binding element (and in particular an ISVD such as a nanobody) that directs a polypeptide of the invention to a particular cell, tissue, or organ

As will be understood by one of skill in the art, when a PD1 binding or LAG3 binding molecule (e.g. containing an ISVD such as a nanobody) of the invention is for systemic administration and/or for the prevention and/or treatment of a chronic disease or disorder, it will generally be preferred, that said PD1 binding or LAG3 binding molecule of the invention has an extended half-life (as described herein). More preferably, such a PD1-binding or LAG3-binding molecule of the invention comprises an ISVD (e.g., nanobody) extending half-life, such as preferably an ISVD, and in particular a human serum albumin-binding nanobody (as described herein).

Some preferred, but non-limiting examples of such a PD1 binding or LAG3 binding molecule of the invention are schematically presented in Tables D-2a and D-2b below, and each is an additional aspect of the invention. Other examples of a suitable PD1 binding or LAG3 binding molecule of the invention with extended half-life will be apparent to the skilled artisan based on the description provided herein. In general, for polypeptides of the invention with an extended half-life, the presence of a C-terminal extension is preferred.

- 49 041987

Table D-2a

Schematic representation of some PD1 and/or LAG3 binding molecules of the invention with increasing half-life (HLE) ISVD [PD-1 binding fragment of the invention]-[HEE]

[HEE] - [PD-1 binding fragment according to the invention]

[PD-1 binding fragment of the invention]-[HLE]-X(n)

I[HEE]-[PD-1 Binding Fragment of the Invention]-X(n)

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[HEE]

[PD-1 binding fragment of the invention]-[HEE]-[Binding

PD-1 fragment according to the invention]

[HEE] - [PD-1 binding fragment according to the invention] - [Binding

PD-1 fragment according to the invention]

[PD-1 binding fragment of the invention]-[PD-1 binding

- 50 041987 fragment according to the invention]-[HLE]-X(n)

[PD-1 binding fragment according to the invention] -[HEE]-[Binding

PD-1 fragment according to the invention] -X(p)

[HEE]-[PD-1 Binding Fragment of the Invention]-[Binding

PD-1 fragment according to the invention] -X(p)

[PD-1 binding fragment according to the invention]-[Another fragment][NE]

[PD-1 Binding Fragment of the Invention] - [PD-1 Binding Fragment of the Invention] - [Other Fragment] - [Other Fragment] [NOT]

[PD-1 Binding Fragment of the Invention]-[Another Fragment][HEE]-X(n)

[PD-1 Binding Fragment of the Invention] - [PD-1 Binding Fragment of the Invention] - [Other Fragment] - [Other Fragment] [HEE] -X(n)

[PD-1 binding fragment according to the invention] - [HER] - [Another fragment]

[HER]-[PD-1 Binding Fragment of the Invention]-[Another Fragment]

[HER] -[Another fragment]-[PD-1 binding fragment according to the invention]

[Other Fragment]-[PD-1 Binding Fragment of the Invention] [NE]

[Another Fragment] -[HEE] -[PD-1 Binding Fragment of the Invention]

[PD-1 binding fragment according to the invention]-[Another fragment][HEE]-X (n)

[PD-1 Binding Fragment of the Invention]-[HEE]-[Other Fragment]-X(η)

[HEE]-[PD-1 Binding Fragment of the Invention]-[Other Fragment]-X (η)

[NE] -[Other fragment] -[PD-1 binding fragment according to the invention] -X(n)

[Other Fragment]-[PD-1 Binding Fragment of the Invention][HEE]-X(n)

- 51 041987

[Another Fragment] -[HEE] -[PD-1 Binding Fragment of the Invention]-X(n)

[PD-1 binding fragment according to the invention]-[Guiding fragment]-[HLE]

[PD-1 Binding Fragment of the Invention]-[HEE]-[Guiding Fragment]

[HEE]-[PD-1 Binding Fragment of the Invention]-[Guide Fragment]

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]-[NEE]

[Guiding Fragment]-[HEE]-[PD-1 Binding Fragment of the Invention]

[HEE]-[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]

[PD-1 binding fragment according to the invention]-[Guiding fragment]-[HEE]-X(η)

[PD-1 binding fragment according to the invention]-[HEE]-[Guiding fragment]-X(η)

[HEE]-[PD-1 Binding Fragment of the Invention]-[Guiding Fragment]-X(η)

[Guiding Fragment]-[PD-1 Binding Fragment of the Invention]-[HEE]-X(n)

[Guiding Fragment]-[HEE]-[PD-1 Binding Fragment of the Invention]-X(n)

[HEE]-[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-X(n)

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[Targeting Fragment]-[HEE]

[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[HEE]-[Guiding Fragment]

[PD-1 binding fragment according to the invention]-[HLE]-[Binding

PD-1 fragment according to the invention]-[Guide fragment]

[HEE]-[PD-1 Binding Fragment of the Invention]-[Binding

PD-1 fragment according to the invention]-[Guide fragment]

[PD-1 binding fragment of the invention]-[PD-lbinder of the

- 52 041987 invention]-[Targeting Fragment]-[HLE]-X(η) [PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[HLE]-[Targeting Fragment]-X ( p) [PD-1 binding fragment of the invention]-[HEE]-[PD-1 binding fragment of the invention]-[Targeting fragment]-X(n)

[HEE]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[Targeting Fragment]-X(n) [Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[Binding PD-1 Fragment of the Invention]-[HEE] [Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[HE]-[PD-1 Binding Fragment of the Invention] [Targeting Fragment]-[HEE]-[Binding PD-1 Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]

[HEE]-[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]

[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[HEE]X(n)

[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[HEE]-[PD-1 Binding Fragment of the Invention]X(n)

[Targeting Fragment]-[HEE]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-X(n)

[HEE]-[Targeting Fragment]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-X(n) [PD-1 Binding Fragment of the Invention]-[HEE]

[HEE]-[PD-1 binding fragment according to the invention]

[PD-1 binding fragment of the invention]-[HEE]-X(n)

[HEE]-[PD-1 Binding Fragment of the Invention]-X(n) [PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]-[HEE]

[PD-1 Binding Fragment of the Invention]-[HEE]-[PD-1 Binding Fragment of the Invention]

[HEE]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention]

- 53 041987

[PD-1 binding fragment of the invention]-[PD-1 binding fragment of the invention]-[HLE]-X(n)

[PD-1 binding fragment of the invention] -[HLE] -[PD-1 binding fragment of the invention] -X(n)

[HEE]-[PD-1 Binding Fragment of the Invention]-[PD-1 Binding Fragment of the Invention] -X(n)

[PD-1 binding fragment according to the invention]-[Another fragment][NE]

Legend:

- "[PD-1 binding fragment of the invention]" represents a PD1 binding domain or binding element, such as PD1ISVD 102C12 (EID, L11V, A14P, A74S, K83R, I89L) described herein

- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker

- “X(n)>> represents a C-terminal extension as described herein, such as a single alanine residue.

- "[HE]" represents a half-life increasing binding domain or binding element (and in particular an ISVD increasing half-life, such as nanobody), such as an ISVD (and in particular, nanobody) to (human) serum albumin, such as anti-HSA ISVD ALB11002 described in this document;

- "[Other Fragment]" represents a binding domain or binding element (eg, ISVD, such as a nanobody) binding an epitope that is different from PD1, for example, one or more of CTLA4-, BTLA-, LAG3-, and/or CD27- An ISVD such as LAG3-ISVD, 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) described herein "[Targeting Fragment]" represents a binding domain or binding element (and in particular an ISVD , such as a nanobody) that target the polypeptide of the invention to a specific cell, tissue, or organ

- 54 041987

Table D-2b

Schematic representation of some PD1 and/or LAG3 binding molecules of the invention with half-life increasing ISVD

[LAG3 binding fragment according to the invention] -[HLE]

[HLE]-[LAG3 binding fragment according to the invention]

[LAG3 binding fragment of the invention]-[HLE]-X(n)

[HEE]-[LAG3 binding fragment of the invention]-X(n)

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[HEE]

[LAG3 binding fragment according to the invention]-[HLE]-[Binding

LAG3 fragment according to the invention]

[HEE]-[LAG3 binding fragment according to the invention]-[Binding

LAG3 fragment according to the invention]

[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-[HLE]-X(n)

[LAG3 binding fragment according to the invention]-[HEE]-[Binding

LAG3 fragment according to the invention] -X(n)

[HEE]-[LAG3 binding fragment according to the invention]-[Binding

LAG3 fragment according to the invention] -X(n)

[LAG3 binding fragment according to the invention]-[Another fragment][NOT]

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Other fragment]-[Another fragment][NE]

[LAG3 binding fragment according to the invention]-[Another fragment] [HEE]-X(n)

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[Other fragment]-[Another fragment][HEE]-X(n)

[LAG3 binding fragment according to the invention] -[HER]-[Another fragment]

[HER]-[LAG3 binding fragment according to the invention]-[Another fragment]

[HER] - [Another fragment] - [LAG3 binding fragment according to the invention]

[Other Fragment]-[LAG3 Binding Fragment of the Invention] [NOT]

- 55 041987

[Another Fragment] -[HER] -[LAG3 Binding Fragment of the Invention]

[LAG3 binding fragment according to the invention]-[Other fragment][HLE]-X (η)

[LAG3 binding fragment according to the invention] -[HEE]-[Another fragment]-X(η)

[HE]-[LAG3 binding fragment according to the invention]-[Another fragment 7-X(η)

[HER] -[Other Fragment]-[LAG3 Binding Fragment of the Invention]-X(n)

[Other Fragment]-[LAG3 Binding Fragment of the Invention] [HEE]-X(n)

[Another Fragment] -[HEE] - [LAG3 Binding Fragment of the Invention]-X(n)

[LAG3 binding fragment according to the invention]-[Guiding fragment]-[HOT]

[LAG3 binding fragment according to the invention]-[HEE]-[Guiding fragment]

[HER]-[LAG3 binding fragment according to the invention]-[Guiding fragment]

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]-[HOT]

[Guiding Fragment]-[HEE]-[LAG3 Binding Fragment of the Invention]

[HEE]-[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]

[LAG3 binding fragment according to the invention]-[Guiding fragment]-[HEE]-X(η)

[LAG3 binding fragment according to the invention]-[HEE]-[Guiding fragment]-X(η)

[HEE] - [LAG3 binding fragment according to the invention] - [Guiding fragment 7-X(η)

[Guiding Fragment]-[LAG3 Binding Fragment of the Invention]-[HEE]-X(n)

[Guiding Fragment]- [HEE]- [Linking LAG3 Fragment by

- 56 041987 of the invention]-X(η) [HLE]-[Targeting fragment]-[LAG3 binding fragment of the invention]-X(n) [LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-[Targeting fragment ]-[HE] [LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[HE]-[Targeting fragment] [LAG3 binding fragment according to the invention]-[HE]-[LAG3 binding fragment according to the invention]-[ Targeting Fragment] [HE]-[LAG3 Binding Fragment of the Invention]-[LAG3 Binding Fragment of the Invention]-[Targeting Fragment] [LAG3 Binding Fragment of the Invention]-[PD-lbinder of the invention]-[Targeting Fragment]-[ HE]-X(n) [LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[HE]-[Targeting fragment]-X(n) [LAG3 binding fragment according to the invention]-[HE]-[Binding Inventive LAG3 Fragment]-[Guiding Fragment]-X(n) [HE]-[Inventive LAG3 Binding Fragment]-[Inventive LAG3 Binding Fragment] of the invention]-[Targeting Fragment]-X(n) [Targeting Fragment]-[LAG3 Binding Fragment of the Invention]-[LAG3 Binding Fragment of the Invention]-[HEE] [Targeting Fragment]-[LAG3 Binding Fragment of the Invention]-[ HE]-[LAG3 binding fragment of the invention] [Targeting fragment]-[HE]-[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention] [HE]-[Targeting fragment]-[LAG3 binding fragment of the invention] - [LAG3 binding fragment of the invention] [Targeting fragment] - [LAG3 binding fragment of the invention] - [LAG3 binding fragment of the invention] - [HEE]X (n) [Targeting fragment] - [LAG3 binding fragment of the invention] - [ HE]-[LAG3-binding fragment of the invention]X(n) [Targeting fragment]-[HE]-[LAG3-binding fragment of the invention]-[LAG3-binding fragment of the invention]-X(n)

- 57 041987

[HLE] - [Guiding Fragment] - [LAG3 Binding Fragment of the Invention] - [LAG3 Binding Fragment of the Invention] -X(n) [LAG3 Binding Fragment of the Invention] - [HLE]

[HEE]-[LAG3 binding fragment according to the invention]

[LAG3 binding fragment of the invention]-[HEE]-X(n) [NEE]-[LAG3 binding fragment of the invention]-X(n)

[LAG3 binding fragment according to the invention]-[LAG3 binding fragment according to the invention]-[HEE]

[LAG3 binding fragment according to the invention]-[HEE]-[LAG3 binding fragment according to the invention]

[HEE]-[LAG3 binding fragment according to the invention]-[ILAG3 binding fragment according to the invention]

[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-[HEE]-X(n)

[LAG3 binding fragment according to the invention] -[HEE]-[Binding

LAG3 fragment according to the invention] -X(p)

[HEE]-[LAG3 binding fragment of the invention]-[LAG3 binding fragment of the invention]-X(n)

[LAG3 binding fragment according to the invention]-[Another fragment][NOT]

Legend:

- "[LAG3 binding fragment according to the invention]" represents a LAG3 binding domain or fragment or element, such as LAG3-ISVD 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R,

V 89L) described in this document

- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker

- "X(n)" represents a C-terminal extension as described herein, such as a single alanine residue.

- "[HE]" represents a half-life increasing binding domain or binding element (and in particular an ISVD increasing half-life, such as nanobody), such as an ISVD (and in particular, nanobody) to (human) serum albumin, such as anti-HSA ISVD ALB11002 described in this document;

- "[Other Fragment]" represents a binding domain or binding element (eg, ISVD, such as a nanobody) binding an epitope that is different from PD1, for example, one or more of CTLA4-, BTLA-, LAG3-, and/or CD27- ISVD such as PD1-ISVD 102C12 (EID, L11V, A14P, A74S, K83R, I89L) described herein

- "[Targeting Fragment]" represents a binding domain or binding element (and in particular an ISVD such as a nanobody) that directs a polypeptide of the invention to a particular cell, tissue, or organ

In an embodiment, the PD1/LAG3 binding molecules of the present invention are shown in Table 1 below. D-3.

- 58 041987

Table D-3

PD1/LAG3 binding molecules of the present invention

Description Subsequence Name: F023700899 Description: 1PD102C12(A14P, A74S, K83R)- 35GS- 1PD102C12(А14Р,A74S,K83R)- 35GS-F0237611B09-35GS- F0237611B09-35GS-ALB11002 Target: hPD-l/hLAG-3 SEQ ID NO: 106 EVOLVESGGGLVOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG RFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGSIASIHAMGWFRQAPGKE RE FVAVITWSGGITYYADSVKGRFTISRDNSKN TVYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSC AASGRTFSDYVMGWFROARGNEREFVAAISESG GRTHYADAVKGRFTISRDNAKNTLYLQMNSLKP EDTAVYYCATTLLWWTSEYAPIKANDYDYWGOG TLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAA SGRTFSDYVMGWFROARGNEREFVAAISESGGR THYADAVKGRFTISRDNAKNTLYLOMNSLKPED TAVYYCATTLLWWTSEYAPIKANDYDYWGOGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGLVQPGNSLRLSCAASG

- 59 041987

FTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSSQGTLVTVSS Name: F023700931 Description: 1PD102C12 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG (EID,LIIV,Al4R,A74S, K83R, I8 9L )-35GS-1PD102C12 (L11V,A14P,A74S,K8 3R,I89L) 35GS-F0237611B09 (LIIV,Al4Р,R4IP,N4ЗК,A62S,А74S,K83R,V89L)-35GS-F0237611В09 (LIIV,Al4Р,R4IP,N4ЗК,A62S,А74S,K83R,V89L)-35GS-ALB11002-A Target: hPD- l/hLAG-3 SEQ ID NO: 107 RETISRDNSKNTVYLQMNSLRPEDTALYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGSIASIHAMGWFROAPGKE RE FVAVITWSGGITYYADSVKGRFTISRDNSKN TVYLOMNSLRPEDTALYYCAGDKHQSSWYDYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGWQPGGSLRLSC AASGRTFSDYVMGWFROAPGKEREFVAAISESG GRTHYADSVKGRFTISRDNSKNTLYLOMNSLRP EDTALYYCATTLLWWTSEYAPIKANDYDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGWQPGGSLRLSCAA SGRTFSDYVMGWFRQAPGKEREFVAAISESGGR THYADSVKGRFTISRDNSKNTLYLQMNSLRPED TALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGNSLRLSCAASG FTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA LYYCTIGGSLSRSSQGTLVTVSSA Name: F023701016 Description : DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG 102C12(E1D,L11V,A14P,A74S,K83 R,I89L)-20GS- 102C12(LIIV,A14P,A74S,K83R,18 9L)-20GS- F0237611B09(LIIV,A14P,R41P,N4 RFTISRDNSKNTVYLQMNSLRPEDTALYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGGSLRLSCAASG SIASIHAMGWFRQAPGKERE FVAVITWSGGITY YADSVKGRFTISRDNSKNTVYLOMNSLRPEDTA 3K,A62S,A74S,K83R,V89L)-20GSF0237611B09(LIIV,A14P,R41P,N4 LYYCAGDKHQSSWYDYWGOGTLVTVSSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGWQPGGSLR

- 60 041987

3K,A62S,A74S,K83R,V89L)-20GS- ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 108 L S CAAS GRTFSDYVMGWFRQAPGKERE FVAAIS ESGGRTHYADSVKGRFTISRDNSKNTLYLQMNS LRPEDTALYYCATTLLWWTSEYAPIKANDYDYW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQ LVESGGGVVOPGGSLRLSCAASGRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFT ISRDNSKNTLYLQMNSLRPEDTALYYCATTLLW WTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGG GGSGGGGSGGGGSEVQLVESGGGWQPGNSLRL S CAAS G FT FS S FGMSWVRQAPGKGLEWVS SIS G SGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTALYYCTIGGSLSRSSQGTLVTVSSA Name: F023701017 Description : 102C12(EID,L11V,A14P,A74S, K83 R, I89L)-9GS- 102C12(LIIV,A14P,A74S,K83R,18 9L)-9GS- F0237611B09(LIIV,Al4P,R4IP,N4 3K,A62S,A74S,K83R,V89L)-9GS- F0237611B09(LIIV,Al4P,R4IP,N4 3K,A62S,A74S,K83R,V89L)-9GS- ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 109 DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG RETISRDNSKNTVYLQMNSLRPEDTALYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGSEVQL VESGGGWQPGGSLRLSCAASGSIASIHAMGWF RQAPGKERE FVAVITWSGGITYYADSVKGRFTI SRDNSKNTVYLOMNSLRPEDTALYYCAGDKHQS SWYDYWGOGTLVTVSSGGGGSGGGSEVOLVESG GGVVOPGGSLRLSCAASGRTFSDYVMGWFRQAP GKERE FVAAISESGGRTHYADSVKGRFTISRDN SKNTLYLOMNSLRPEDTALYYCATTLLWWTSEY APIKANDYDYWGOGTLVTVSSGGGGSGGGSEVQ LVESGGGVVOPGGSLRLSCAASGRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFT ISRDNSKNTLYLOMNSLRPEDTALYYCATTLLW WTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGG GSEVQLVESGGGWQPGNSLRLSCAASGFTFSS FGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV KGRFTISRDNAKTTLYLQMNSLRPEDTALYYCT IGGSLSRSSQGTLVTVSSA Name: F023700924 Description: 1PD102C12 (EID,LIIV,Al4P,A74S, K83R, I8 9L DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG RFTISRDNSKNTVYLQMNSLRPEDTALYYCAGD

- 61 041987

)-35GS- F0237611B09 (L11V,A14P,R41P,N4 3K,A62S, A7 4 S,K83R,V89L)-35GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 110 KHQSSWYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGRTFSDYVMGWFROAPGKE RE FVAAISESGGRTHYADSVKGRFTISRDNSKN TLYLOMNSLRPEDTALYYCATTLLWWTSEYAPI KANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGWQ PGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVT VS SA Name: F023700969 Description : 102C12(EID,LIIV,Al4P,A74S,K83 R, I89L)-20GS- F0237611B09(LIIV,A14P,R41P,N4 3K,A62S,A74S,K83R,V89L)-20GSALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 111 DVOLVESGGGVVOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG RFTISRDNSKNTVYLQMNSLRPEDTALYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGGSLRLSCAASG RTFSDYVMGWFRQAPGKEREFVAAISESGGRTH YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTA LYYCATTLLWWTSEYAPIKANDYDYWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGG WQPGNSLRLSCAASGFTFSSFGMSWVRQAPGK GLEWVSSISGSGSDTLYADSVKGRFTISRDNAK TTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGT LVTVSSA Name: F023700970 Description: 102C12 (E1D,L11V,A14P,A7 4S,K8 3R, I8 9L )-9GS-F0237611B09 (L11V,A14P,R41P,N4 3K, A62S, A7 4 S,K83R,V89L)-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 112 DVOLVESGGGVVOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITWSGGITYYADSVKG RFTISRDNSKNTVYLQMNSLRPEDTALYYCAGD KHQSSWYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGRTFSDYVMGWF RQAPGKERE FVAAISESGGRTHYADSVKGRFTI SRDNSKNTLYLOMNSLRPEDTALYYCATTLLWW TSEYAPIKANDYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGWQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTALYYCTI

- 62 041987

GGSLSRSSSQGTLVTVSSA Name: F023701163 Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)- 35GS-1PD102C12 (L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)-35GS- F023700842-35GS-F02370084235GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 113 DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDPSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGSIASIHAMGWFROAPGKE RE FVAVITVSGGITYYADSVKGRFTISRDPSKN TVYLOMNSLRPEDTALYYCAGDKHQSSFYDYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGWQPGGSLRLSC AASGRTFSDYVMGWFROAPGKEREFVAAISESG GRTHYADSVKGRFTISRDNSKNTLYLOMNSLRP EDTALYYCATTLLWWTSEYAPIKANDYDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGWQPGGSLRLSCAA SGRTFSDYVMGWFRQAPGKEREFVAAISESGGR THYADSVKGRFTISRDNSKNTLYLOMNSLRPED TALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGNSLRLSCAASG FTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA LYYCTIGGSLSRSSQGTLVTVSSA Name: F023701168 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)- 9GS-1PD102C12 (L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)-9GS-F0237008429GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 114 DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDPSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGSIASIHAMGWF RQAPGKERE FVAVITVSGGITYYADSVKGRFTI SRDPSKNTVYLOMNSLRPEDTALYYCAGDKHQS SFYDYWGOGTLVTVSSGGGGSGGGSEVOLVESG GGWOPGGSLRLSCAASGRTFSDYVMGW FRQAP GKERE FVAAISESGGRTHYADSVKGRFTISRDN SKNTLYLOMNSLRPEDTALYYCATTLLWWTSEY

- 63 041987

APIKANDYDYWGQGTLVTVSSGGGGSGGGSEVQ LVE S GGGWQPGGS LRLS CAASGRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFT ISRDNSKNTLYLOMNSLRPEDTALYYCATTLLW WTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGG GSEVQLVESGGGWQPGNSLRLSCAASGFTFSS FGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV KGRFTISRDNAKTTLYLQMNSLRPEDTALYYCT IGGSLSRSSQGTLVTVSSA Name: F023701173 Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)- 35GS-F023700842-35GS- ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 115 DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDPSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGRTFSDYVMGWFROAPGKE RE FVAAISESGGRTHYADSVKGRFTISRDNSKN TLYLQMNSLRPEDTALYYCATTLLWWTSEYAPI KANDYDYWGOGTLVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGWQ PGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVT VS SA Name: F023701178 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73P, A74S, K83R, I89L, WIOOaF)- 9GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 116 DVQLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDPSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGRTFSDYVMGWF RQAPGKERE FVAAISESGGRTHYADSVKGRFTI SRDNSKNTLYLQMNSLRPEDTALYYCATTLLWW TSEYAPIKANDYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGWQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTALYYCTI GGSLSRSSQGTLVTVSSA

- 64 041987

Name: F023701161 Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WIOOaF)- 35GS-1PD102C12 (L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WIOOaF)-35GS- F023700842-35GS-F02370084235GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 117 DVOLVESGGGWOPGGSLRLSCAASGSIASIHA MGW FRQAP GKE RE FVAVITVSGGITYYADSVKG RFTISRDQSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGSIASIHAMGWFROAPGKE RE FVAVITVSGGITYYADSVKGRFTISRDQSKN TVYLOMNSLRPEDTALYYCAGDKHQSSFYDYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGWQPGGSLRLSC AASGRTFSDYVMGWFRQAPGKERE FVAAISESG GRTHYADSVKGRFTISRDNSKNTLYLOMNSLRP EDTALYYCATTLLWWTSEYAPIKANDYDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGWQPGGSLRLSCAA SGRTFSDYVMGWFRQAPGKEREFVAAISESGGR THYADSVKGRFTISRDNSKNTLYLQMNSLRPED TALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSSGGGGSGGGGSGGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGNSLRLSCAASG FTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA LYYCTIGGSLSRSSQGTLVTVSSA Name: F023701166 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WIOOaF)- 9GS-1PD102C12 (L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WIOOaF)-9GS-F0237008429GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 118 DVQLVESGGGWOPGGSLRLSCAASGSIASIHA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDQSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGSIASIHAMGWF RQAPGKERE FVAVITVSGGITYYADSVKGRFTI SRDOSKNTVYLOMNSLRPEDTALYYCAGDKHQS SFYDYWGOGTLVTVSSGGGGSGGGSEVOLVESG GGVVOPGGSLRLSCAASGRTFSDYVMGWFRQAP GKERE FVAAISESGGRTHYADSVKGRFTISRDN SKNTLYLQMNSLRPEDTALYYCATTLLWWTSEY APIKANDYDYWGQGTLVTVSSGGGGSGGGSEVQ

- 65 041987

LVESGGGWOPGGSLRLSCAASGRTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKGRFT ISRDNSKNTLYLOMNSLRPEDTALYYCATTLLW WTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGG GSEVQLVESGGGWQPGNSLRLSCAASGFTFSS FGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV KGRFTISRDNAKTTLYLQMNSLRPEDTALYYCT IGGSLSRSSQGTLVTVSSA Name: F023701171 Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WlOOaF)- 35GS-F023700842-35GS- ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 119 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RETISRDQSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGRTFSDYVMGWFROAPGKE RE FVAAISESGGRTHYADSVKGRFTISRDNSKN TLYLQMNSLRPEDTALYYCATTLLWWTSEYAPI KANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGWQ PGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVT VS SA Name: F023701176 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73Q, A74S, K83R, I89L, WlOOaF)- 9GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 120 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDQSKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWQPGGSLRLSCAASGRTFSDYVMGWF RQAPGKERE FVAAISESGGRTHYADSVKGRFTI SRDNSKNTLYLOMNSLRPEDTALYYCATTLLWW TSEYAPIKANDYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGWQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTALYYCTI GGSLSRSSQGTLVTVSSA Name: F023701162 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA

- 66 041987

Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WIOOaF)- 35GS-1PD102C12 (L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WIOOaF)-35GS- F023700842-35GS-F02370084235GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 121 MGWFRQAP GKERE FVAVITVSGGITYYADSVKG RFTISRDS SKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGSIASIHAMGWFROAPGKE RE FVAVITVSGGITYYADSVKGRFTISRDS SKN TVYLOMNSLRPEDTALYYCAGDKHQSSFYDYWG QGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGWQPGGSLRLSC AASGRTFSDYVMGWFROAPGKEREFVAAISESG GRTHYADSVKGRFTISRDNSKNTLYLOMNSLRP E D TALYYCATTLLWWTSEYAPIKANDYDYWGQG TLVTVSSGGGGSGGGGGSGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGWQPGGSLRLSCAA SGRTFSDYVMGWFRQAPGKEREFVAAISESGGR THYADSVKGRFTISRDNSKNTLYLQMNSLRPED TALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGWQPGNSLRLSCAASG FTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA LYYCTIGGSLSRSSQGTLVTVSSA Name: F023701167 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WIOOaF)- 9GS-1PD102C12 (L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WIOOaF)-9GS-F0237008429GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 122 DVOLVESGGGVVOPGGSLRLSCAASGSIASIHA MGWFRQAP GKERE FVAVITVSGGITYYADSVKG RFTISRDS SKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGSIASIHAMGWF RQAPGKERE FVAVITVSGGITYYADSVKGRFTI SRDSSKNTVYLOMNSLRPEDTALYYCAGDKHQS SFYDYWGOGTLVTVSSGGGGSGGGSEVQLVESG GGWOPGGSLRLSCAASGRTFSDYVMGW FRQAP GKERE FVAAISESGGRTHYADSVKGRFTISRDN SKNTLYLOMNSLRPEDTALYYCATTLLWWTSEY APIKANDYDYWGQGTLVTVSSGGGGSGGGSEVQ LVESGGGWOPGGSLRLSCAASGRTFSDYVMGW

- 67 041987

FRQAPGKERE FVAAISESGGRTHYADSVKGRFT ISRDNSKNTLYLOMNSLRPEDTALYYCATTLLW WTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGG GSEVQLVESGGGWQPGNSLRLSCAASGFTFSS FGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV KGRFTISRDNAKTTGSLYLQMNSLRPEDTALYYQ IGGSATRS

Name: F023701172 Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WlOOaF)- 35GS-F023700842-35GS- ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 123 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RETISRDS SKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGV VOPGGSLRLSCAASGRTFSDYVMGWFROAPGKE RE FVAAISESGGRTHYADSVKGRFTISRDNSKN TLYLQMNSLRPEDTALYYCATTLLWWTSEYAPI KANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGWQ PGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE WVSSISGSGSDTLYADSVKGRFTISRDNAKTTL YLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVT VS SA Name: F023701177 Description: 1PD102C12 (EID, L11V, A14P, W52aV, N73S, A74S, K83R, I89L, WlOOaF)- 9GS-F023700842-9GS-ALB11002-A Target: hPD-l/hLAG-3 SEQ ID NO: 124 DVQLVE S GGGWQPGGS LRL S CAAS GS IAS I HA MGWFRQAPGKERE FVAVITVSGGITYYADSVKG RFTISRDS SKNTVYLQMNSLRPEDTALYYCAGD KHQSSFYDYWGOGTLVTVSSGGGGSGGGSEVOL VESGGGWOPGGSLRLSCAASGRTFSDYVMGWF RQAPGKERE FVAAISESGGRTHYADSVKGRFTI SRDNSKNTLYLOMNSLRPEDTALYYCATTLLWW TSEYAPIKANDYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGWQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTALYYCTI GGSLSRSSQGTLVTVSSA

*CDRs of PD1 binding molecules and LAG3 binding molecules are underlined and/or in bold

Optionally, the first residue of any binding fragment of the molecule is substituted with D or E, if necessary.

The present invention includes any PD1/LAG3 binding molecule containing, the amino acid sequence of SEQ ID NO: 106-124, or an amino acid sequence having 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99%) amino acid sequence identity (ie when comparing full length amino acid sequences), where the PD1/LAG3 binding molecule retains the ability to bind to PD1 and LAG3, and optionally HSA.

The present invention includes any PD1/LAG3 binding molecule having the following fragment arrangement.

- 68 041987

- PD1 binding fragment 1PD102C12 (A14P, A74S, K83R) -

- Peptide linker, e.g. 35GS-

- PD1 binding fragment 1PD102C12 (A14P, A74S, K83R) -

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F0237611B09-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F0237611B09-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002 or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 35GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 35GS-

- 69 041987

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 102C12 (EID, L11V, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 2 0GS-

- PD1 binding fragment

102С12(L11V,А14Р,A74S,K83R,I89L)-

- Peptide linker, e.g. 2 0GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 2 0GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 2 0GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 102C12 (EID, L11V, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 9GS-

- PD1 binding fragment 102C12 (L11V, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- 70 041987

- PD1 binding fragment 1PD102C12 (EID, LIIV, Al4P, A 74S, K83R, I89L) -

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F0237611B09 (LIIV, Al4P, R4IP, N4ZK, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment

102C12 (EID, LIIV, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 2 0GS-

- LAG3 binding fragment

F0237611B09(LIIV,Al4P,R4IP,N4ZK,A62S,A74S,K83R,V89L)-

- Peptide linker, e.g. 2 0GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 102C12 (EID, LIIV, A14P, A74S, K83R, I89L) -

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F0237611B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) -

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- 71 041987

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73P, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- 72 041987

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- 73 041987

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73Q, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73S, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73S, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73S, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- PD1 binding fragment 1PD102C12 (L11V, A14P, W52aV,

N73S, A74S, K83R, I89L, WlOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- 74 041987

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73S, A74S, K83R, I89L, WIOOaF)-

- Peptide linker, e.g. 35GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 35GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine or

- PD1 binding fragment 1PD102C12 (EID, L11V, A14P,

W52aV, N73S, A74S, K83R, I89L, WIOOaF)-

- Peptide linker, e.g. 9GS-

- LAG3 binding fragment F023700842-

- Peptide linker, e.g. 9GS-

- Human serum albumin binding fragment, e.g. ALB11002-

- Optional C-terminal extension alanine.

In an embodiment, the PD1/LAG3 binding fragments of the present invention include the following fragment arrangement:

- PD1 binding fragment SEQ ID NO: 98;

- Linker 35GS SEQ ID NO: 58;

- PD1 binding fragment SEQ ID NO: 98;

- Linker 35GS SEQ ID NO: 58;

- Binding LAG3 fragment SEQ ID NO: 63;

- Linker 35GS SEQ ID NO: 58;

- Binding LAG3 fragment SEQ ID NO: 63;

- Linker 35GS SEQ ID NO: 58;

- HSA binding fragment SEQ ID NO: 59;

- Alanine

- 75 041987 or

- PD1 binding fragment SEQ ID NO: 57; - Linker 35GS SEQ ID NO: 58; - PD1 binding fragment SEQ ID NO: 99; - Linker 35GS SEQ ID NO: 58; - LAG3 binding fragment SEQ ID NO: :64 - Linker 35GS SEQ ID NO: 58; - LAG3 binding fragment SEQ ID NO: :64 - Linker 35GS SEQ ID NO: 58; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment SEQ ID NO: 57; - Linker 20GS SEQ ID NO: 100; - PD1 binding fragment SEQ ID NO: 99; - Linker 20GS SEQ ID NO: 100; - LAG3 binding fragment SEQ ID NO: :64 - Linker 20GS SEQ ID NO: 100; - LAG3 binding fragment SEQ ID NO: :64 - Linker 20GS SEQ ID NO: 100; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment SEQ ID NO: 57; - Linker 9GS SEQ ID NO: 125; - PD1 binding fragment SEQ ID NO: 99; - Linker 9GS SEQ ID NO: 125; - LAG3 binding fragment SEQ ID NO: :64 - Linker 9GS SEQ ID NO: 125; - LAG3 binding fragment SEQ ID NO: :64 - Linker 9GS SEQ ID NO: 125; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment SEQ ID NO: 57;

Linker 35GS SEQ ID NO: 58

- 76 041987

- LAG3 binding fragment SEQ ID NO: 64

- Linker 35GS SEQ ID NO: 58;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment SEQ ID NO: 57;

- Linker 20GS SEQ ID NO: 100;

- LAGS binding fragment SEQ ID NO: 64

- Linker 20GS SEQ ID NO: 100;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

PD1 binding fragment SEQ ID NO: 57;

Linker 9GS SEQ ID NO: 125;

LAGS binding fragment SEQ ID NO: 64

- Linker 9GS SEQ ID NO: 125; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment SEQ ID NO: 105; - Linker 35GS SEQ ID NO: 58; - PD1 binding fragment of SEQ ID NO: 105 (DIE); - Linker 35GS SEQ ID NO: 58;

LAGS binding fragment SEQ ID NO: 64

- Linker 35GS SEQ ID NO: 58; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment SEQ ID NO: 105; - Linker 9GS SEQ ID NO: 125; - PD1 binding fragment of SEQ ID NO: 105 (DIE); - Linker 9GS SEQ ID NO: 125; - LAGS binding fragment SEQ ID NO: 64; - Linker 9GS SEQ ID NO: 125; - HSA binding fragment SEQ ID NO: 59;

Alanine

- 77 041987 or

- PD1 binding fragment SEQ ID NO: 105;

- Linker 35GS SEQ ID NO: 58;

- LAGS binding fragment SEQ ID NO: 64;

- Linker 35GS SEQ ID NO: 58;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment SEQ ID NO: 105;

- Linker 9GS SEQ ID NO: 125;

- LAGS binding fragment SEQ ID NO: 64;

- Linker 9GS SEQ ID NO: 125;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment SEQ ID NO: 104;

- Linker 35GS SEQ ID NO: 58;

- PD1 binding fragment SEQ ID NO: 104 (DIE)

- Linker 35GS SEQ ID NO: 58;

- LAGS binding fragment SEQ ID NO: 64;

- Linker 35GS SEQ ID NO: 58; - Binding HSA fragment SEQ ID NO: 59; - Alanine or - Binding PD1 fragment SEQ ID NO: 104; - Linker 9GS SEQ ID NO: 125; - Binding PD1 fragment of SEQ ID NO: 104 (DIE); - Linker 9GS SEQ ID NO: 125;

LAGS binding fragment SEQ ID NO: 64

Linker 9GS SEQ ID NO: 125;

HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment SEQ ID NO: 104

- Linker 35GS SEQ ID NO: 58;

- LAGS binding fragment SEQ ID NO: 64

- 78 041987

- Linker 35GS SEQ ID NO: 58;

- HSA binding fragment SEQ ID NO: 59

- Alanine or

PD1 binding fragment SEQ ID NO: 104

Linker 9GS SEQ ID NO: 125;

LAGS binding fragment SEQ ID NO: 64

- Linker 9GS SEQ ID NO: 125; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment of SEQ ID NO: 103 (N73S); - Linker 35GS SEQ ID NO: 58; - PD1 binding fragment of SEQ ID NO:103 (DIE, N73S); - Linker 35GS SEQ ID NO: 58;

LAGS binding fragment SEQ ID NO: 64

- Linker 35GS SEQ ID NO: 58; - HSA binding fragment SEQ ID NO: 59; - Alanine or - PD1 binding fragment of SEQ ID NO: 103 (N73S); - Linker 9GS SEQ ID NO: 125; - PD1 binding fragment of SEQ ID NO:103 (DIE, N73S); - Linker 9GS SEQ ID NO: 125;

LAGS binding fragment SEQ ID NO: 64

- Linker 9GS SEQ ID NO: 125;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment of SEQ ID NO: 103 (N73S);

- Linker 35GS SEQ ID NO: 58;

- LAG3 binding fragment SEQ ID NO: 64;

- Linker 35GS SEQ ID NO: 58;

- HSA binding fragment SEQ ID NO: 59;

- Alanine or

- PD1 binding fragment of SEQ ID NO: 103 (N73S);

- Linker 9GS SEQ ID NO: 125;

- LAGS binding fragment SEQ ID NO: 64;

- Linker 9GS SEQ ID NO: 125;

- HSA binding fragment SEQ ID NO: 59;

- Alanine

Cross-blocking antibodies.

Настоящее изобретение также относится к перекрестно-блокирующим связывающим молекулам, которые способны перекрестно блокировать связывание любой из связывающих молекул, описанных в настоящем документе (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176 ; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177). Such

- 79 041987 cross-blocking binding molecules can be any molecule that exhibits such cross-blocking properties, for example, ISVD, nanobody, antibody or antigen-binding fragment.

In general, a binding molecule (e.g., an ISVD, such as a nanobody) or an antibody, or an antigen-binding fragment thereof, that cross-blocks a reference binding molecule or cross-competes with a reference binding molecule refers to a binding molecule (e.g., an ISVD, such as a nanobody) or antibody, or an antigen-binding fragment thereof that block the binding of a reference binding molecule to its antigen in a competitive assay by 50% or more, and conversely, the reference binding molecule blocks the binding of a binding molecule (e.g., an ISVD such as a nanobody) or an antibody, or an antigen-binding fragment thereof with their antigen in a competitive assay by 50% or more. Cross-blocking or cross-competition can be determined by any assay known in the art, including surface plasmon resonance (SPR), ELISA, and flow cytometry.

In an embodiment of the invention, cross-blocking is determined using a Biacore assay. For convenience, reference is made to two binding molecules, the scope of the present invention includes antibodies and their antigen-binding fragments, such as Fab fragments, which cross-block the binding molecule of the present invention. Operations on the Biacore instrument (eg Biacore 3000) are carried out in accordance with the manufacturer's recommendations.

Thus, in one cross-blocking assay, PD1 or LAG3 is coupled to a CM5 Biacore chip using standard amino chemistries to produce a surface coated with PD1 or LAG3. For example, 200-800 resonant units of PD1 or LAG3 are bound to the chip (or any amount that gives easily measurable levels of binding, but is easily saturable with the concentrations of the test reagent used).

The two binding molecules (called A* and B*) to be assessed for their ability to cross-block each other are mixed in a one to one molar ratio of binding sites in an appropriate buffer to create a test mixture.

The concentration of each binding molecule in the test mixture should be high enough to readily saturate the binding sites of that binding molecule on the PD1 or LAG3 molecules captured on the Biacore chip. The binding molecules in the mixture have the same molar concentration.

Separate solutions are also prepared containing only the binding molecule A* and only the binding molecule B*. Binding molecule A* and binding molecule B in these solutions must be in the same buffer and at the same concentration as in the test mixture.

The test mixture is passed through a Biacore chip coated with PD1 or LAG3 and the overall binding is recorded. The chip is then processed in such a way as to remove bound binding molecules without damaging chip-bound PD1 or LAG3. In an embodiment of the invention, this is done by treating the chip with 30 mM HCl for 60 seconds.

Then, a solution of only the binding molecule A* is passed through the surface coated with PD1 or LAG3 and the degree of binding is recorded. The chip is processed again to remove all associated binding molecules without damaging the PD1 or LAG3 associated with the chip.

Then, a solution of only the binding molecule B* is passed through the surface coated with PD1 or LAG3 and the degree of binding is recorded.

The maximum theoretical binding of the mixture of binding molecule A* and binding molecule B* is then calculated and is the sum of the binding of each binding molecule when passing through a surface coated with PD1 or LAG3. If the actual binding of the mixture is below this theoretical maximum, then the two binding molecules cross block each other.

Thus, in general, a cross-blocking binding molecule of the invention is a molecule that will bind to PD1 or LAG3 in the above Biacore cross-blocking assay, such that during the assay and in the presence of a second binding molecule, the detectable binding is, for example, from 80 % to 0.1% (e.g. 80% to 4%) of maximum theoretical binding, e.g. 75% to 0.1% (e.g. 75% to 4%) of maximum theoretical binding, e.g. 70 % to 0.1% (eg, 70% to 4%) of the maximum theoretical binding (as defined above) of the two binding molecules in combination.

In one embodiment of the invention, an ELISA assay is used to determine if the PD1 and/or LAG3 binding molecules are cross-blocking or capable of cross-blocking in accordance with the invention.

The general principle of the assay is to coat the wells of an ELISA plate with PD1 or LAG3 binding molecules. An excess amount of a second potentially cross-blocking anti-PD1 or LAG3 binding molecule (ie, not bound to the ELISA plate) is added to the solution. A limited amount of PD1 or LAG3 is then added to the wells. Immobilized binding molecules and binding molecules in solution compete for binding a limited

- 80 041987 number of PD1 or LAG3 molecules. The plate is washed to remove PD1 or LAG3 that is not bound by the immobilized binding molecules, and also remove the second binding molecules in solution, as well as any complexes formed between the second binding molecules in solution and PD1 or LAG3. The amount of bound PD1 or LAG3 is then measured using the appropriate PD1 or LAG3 detection reagent. Binding molecules in solution that can cross-block immobilized binding molecules on the surface of the wells can result in a decrease in the number of PD1 or LAG3 molecules that the surface-immobilized molecules can bind relative to the number of PD1 or LAG3 molecules that the surface-immobilized molecules can bind in the absence of the second binding molecules in solution.

expression methods.

Настоящее изобретение включает рекомбинантные способы получения связывающих PD1 и/или LAG3 молекул (например, ISVD, таких как нанотела) по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176 ; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) comprising (i) introducing a polynucleotide encoding the amino acid sequence of said binding PD1 and/or LAG3 molecule, for example, where is located and/or functional polynucleotide/molecule in the vector associated with a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for the expression of the polynucleotide, and (iii) optionally isolating PD1 and/or LAG3 binding molecules from the host cell and/or the medium in which the host cell. See, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to polynucleotides that encode PD1 and/or LAG3 binding molecules of the present invention (e.g. ISVDs such as nanobodies) described herein (e.g. ; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701170 or F0237). Polynucleotides in an embodiment of the invention may be operably linked to one or more regulatory sequences. The polynucleotide may be in the form of a plasmid or a vector. In addition, such polynucleotides may be generally as described in Ablynx N.V. published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to hosts or host cells that contain such polynucleotides encoding PD1 and/or LAG3 binding molecules, vectors and/or PD1 and/or LAG3 binding polypeptide described herein (for example, F023700899; F023700931; F023701016; F023701017 ; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171, F023701176, F023701162, F023701167, F023701172 или F023701177). In addition, such host cells may generally be as described in Ablynx N.V. published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627. Examples of specific host cells are discussed below.

Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expressing a PD1 and/or LAG3 binding molecule (e.g., ISVD, such as a nanobody) are well known in the art and include many immortalized cell lines available from the American Type Collection. cultures (ATSS). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma (e.g. Hep G2) cells, A549, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells.

Preferred cell lines are selected by determining which cell lines have high levels of expression. Other cell lines that can be used are insect cell lines (eg Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast cells and filamentous fungi, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Fusarium spnowense, Chrysosporium luck ., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica and Neurospora crassa. The present invention includes any host cell (for example, a cell

- 81 041987

СНО или клетку Pichia, например Pichia pastoris), содержащую связывающую PD1 и/или LAG3 молекулу по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161 ; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) or containing a polynucleotide encoding such a binding molecule, or containing a vector that contains a polynucleotide.

In addition, the expression of a PD1 and/or LAG3 binding molecule (eg, ISVD, such as a nanobody) in production cell lines can be enhanced using a number of known methods. For example, the glutamine synthetase gene expression system (GS-system) is a standard approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in European Patent Nos. 0216846, 0256055 and 0323997 and in European Patent Application No. 89303964.4. Thus, in one embodiment of the invention, mammalian host cells (e.g., CHO) lack the glutamine synthetase gene and are grown in the absence of glutamine in a medium where, however, the polynucleotide encoding the immunoglobulin chain contains the glutamine synthetase gene, which makes up for the absence of the gene in the host cell. . Such host cells containing the binding molecule or polynucleotide or vector discussed herein, as well as the expression methods discussed herein to produce a binding molecule using such a host cell, are part of the present invention.

Настоящее изобретение включает способы очистки связывающей PD1 и/или LAG3 молекулы (например, ISVD, такого как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161 ; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) involving the introduction of a sample (e.g. culture medium, cell lysate or cell lysate fraction, e.g. soluble lysate fraction) containing PD1 and/or LAG3 binding molecules eg cation exchange medium, anion exchange medium and/or hydrophobic exchange medium) and either collection of purified PD1 and/or LAG3 binding molecules from the breakthrough fraction for said sample that do not bind to the medium; or by discarding the breakthrough fraction, the bound PD1 and/or LAG3 binding molecules are eluted from the medium and the eluate is collected. In an embodiment of the invention, the medium is in the column into which the sample is applied. In an embodiment of the invention, purification is carried out after recombinant expression of the antibody or fragment in a host cell, for example, where the host cell is first lysed and, optionally, the lysate is purified from insoluble materials before purification on the medium; or wherein the PD1 and/or LAG3 binding molecules are secreted into the culture medium by the host cell and the medium or fraction thereof is applied to the purification medium.

In general, glycoproteins produced in a particular cell line or transgenic animal will have glycosylation that is characteristic of glycoproteins produced in that cell line or that transgenic animal. Поэтому конкретные позиции гликозилирования связывающей PD1 и/или LAG3 молекулы (например, ISVD, такого как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) will depend on the particular cell line or transgenic animal used to generate the PD1 and/or LAG3 binding molecule. PD1 and/or LAG3 containing only non-fucosylated N-glycans are part of the present invention and may be useful because non-fucosylated antibodies have been shown to be more effective than their fucosylated counterparts both in vitro and in vivo (see for example, Shinkawa et al., J Biol Chem 278: 3466-3473 (2003) US Pat. These PD1 and/or LAG3 binding molecules with non-fucosylated N-glycans are unlikely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

The present invention includes PD1 and/or LAG3 binding molecules (e.g., ISVDs such as nanobodies) comprising N-linked glycans that are typically added to immunoglobulins produced in Chinese Hamster Ovary cells (CHO cell N-linked glycans) or in modified yeast cells (N-linked glycans of engineered yeast), such as, for example, Pichia pastoris. For example, in an embodiment, the PD1 and/or LAG3 binding molecule comprises one or more genetically engineered yeast N-linked glycans or CHO cell N-linked glycans as shown in FIG. 4 (eg G0 and/or G0-F and/or G1 and/or G1-F and/or G2-F and/or Man5). In an embodiment, the PD1 and/or LAG3 binding molecule comprises N-linked glycans of engineered yeast, namely G0 and/or G1 and/or G2, optionally further including Man5. In an embodiment, the PD1 and/or LAG3 binding molecule comprises N-linked CHO cell glycans, namely G0-F, G1-F and G2-F, optionally further including G0 and/or G1 and/or G2, and /or Man5. In an embodiment of the invention from 80 to 95%

- 82 041987 (for example, about 80-90%, about 85%, about 90%, or about 95%) of all N-linked glycans on PD1 and/or LAG3 binding molecules are N-linked glycans of genetically modified yeast or N-linked glycans of CHO cells. See Nett et al. Yeast. 28(3): 237-252 (2011); Hamilton et al. Science. 313(5792): 1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in an embodiment, the engineered yeast cell is GFI5.0 or YGLY8316 or the strains described in US Pat. No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. WO2013/066765.

Combinations.

В конкретных вариантах осуществления, связывающие PD1 и/или LAG3 молекулы (например, ISVD, такие как нанотела) по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) may be used alone or in conjunction with other, additional therapeutic agents and/or therapeutic procedures for the treatment or prevention of any disease, such as the present, for example, cancer discussed. document, in a subject in need of such treatment or prophylaxis. Compositions or kits, such as pharmaceutical compositions, containing a pharmaceutically acceptable carrier containing such PD1 and/or LAG3 binding molecules in combination with other therapeutic agents, are also part of the present invention.

Термин связанные означает, что компоненты, связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172; or several compositions (for example, in a set). Each component may be administered to the subject at a different time relative to the administration of the other component; for example, each administration may be non-simultaneous (eg, separate or sequential) at intervals over a predetermined period of time. In addition, the individual components may be administered to the subject by one or more routes (eg, if the PD1 and/or LAG3 binding molecule of the present invention is administered parenterally and paclitaxel is administered orally).

В конкретных вариантах осуществления связывающие PD1 и/или LAG3 молекулы (например, ISVD, такое как нанотела) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) may be used in conjunction with an anticancer therapeutic agent or with an immunomodulatory drug such as an immunomodulatory receptor inhibitor, for example an antibody or antigen-binding fragment thereof that binds specifically to a receptor.

В варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172; platinum agent, BRAF inhibitor, CDK4/6 inhibitor, EGFR inhibitor, VEGF inhibitor, microtubule stabilizer, taxane, CD20 inhibitor, CD52 inhibitor, CD30 inhibitor, RANK inhibitor activating nuclear factor kappa-B), ERK inhibitor, MAP kinase inhibitor, AKT inhibitor, MEK inhibitor, PI3K inhibitor, ing a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a Bc12 inhibitor, a CD22 inhibitor, a CD79b inhibitor, an ErbB2 inhibitor, or a farnesyl transferase inhibitor.

В варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is associated with one or more anti-PD1 antibodies or their antigen-binding fragment (for example, pembrolizumab, nivolumab, CT-011, anti-TIM3, anti-TIM3 , anti-CS1 (eg, elotuzumab), anti-KIR2DL1/2/3 (eg, lirilumab), anti-CD27, anti-CD137 (eg, urelumab) GITR (eg, TRX518), anti-PD-L1 (eg, BMS-936559, MSB0010718C or MPDL3280A), anti-PD-L2, anti-ILT1, anti-[LT2, anti-[LT3, anti-[LT4, anti-ILT5, anti-ILT6, anti-[LT7, anti-[ Lτ8, anti-CD40, anti-OX40, anti-CD137, anti-KIR2DLl, anti-KIR2DL2/3, anti-KIR2DL4, anti-KIR2DL5A,

- 83 041987 aHTu-KIR2DL5B, aHTu-KIR3DL1, aHTu-KIR3DL2, aHTu-KIR3DL3, aHTu-NKG2A, aHTu-NKG2C, antiNKG2E or any low molecular weight organic inhibitor of such targets; IL-10, anti-ILlO, anti-TSLP (thymus-stromal lymphopoietin), or PEGylated IL-10.

In an embodiment of the invention, the molecular weight of the polyethylene glycol (PEG) moiety on the PEGylated IL-10 molecule is about 12,000 daltons, or about 20,000 daltons. In an embodiment, PEGylated IL-10 (e.g., PEGylated human IL-10) comprises one or more polyethylene glycol molecules covalently linked via a linker (e.g., C ₂ _ ₁₂ -alkyl such as -CH2CH2CH2-) to one amino acid residue of one subunit IL-10, wherein said amino acid residue is an alpha-amino group of an N-terminal amino acid residue or an epsilon-amino group of a lysine residue. In an embodiment of the invention PEGylated IL-10 is: (PEG) _b -L-NH-IL-10; where b is 1-9 and L is a C ₂ _ ₁₂ alkyl linker moiety covalently attached to the nitrogen (N) of one amino acid residue of IL-10. In an embodiment of the invention PEGylated IL-10 has the formula: [X--O(CH ₂ CH ₂ O) _n ] _b -L-NH-IL-10, where X is H or C ₁ . ₄ -alkyl; n is from 20 to 2300; b is from 1 to 9; and L is C ₁ . ₁₁ -alkyl linker fragment, which is covalently attached to the nitrogen (N) of the alpha-amino group at the amino terminus of one subunit of IL-10; provided that when b is greater than 1, the total number of n does not exceed 2300. See US 7,052,686.

In an embodiment, the anti-IL-10 antibody or antigen-binding fragment thereof (eg, a humanized antibody) contains the CDRs below:

CDR-L1 CDR-L2 CDR-L3 CDR-H1 CDR-H2 CDR-H3 KTSQNIFENLA (SEQ ID NO: 71)

NASPLQA (SEQ ID NO: 72) HQYYSGYT (SEQ ID NO: 73) GFTFSDYHMA (SEQ ID NO: 74) SITLDATYTYYRDSVRG (SEQ ID NO: 75) HRGFSVWLDY (SEQ ID NO: ID NO: 76) (see US 7662379).

In an embodiment, the anti-TSLP antibody or antigen-binding fragment thereof (eg, a humanized antibody) contains the CDRs below:

CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 GYIFTDYAMH (SEQ ID NO: 77)

TFIPLLDTSDYNQNFK (SEQ ID NO: 78; MGVTHSYVMDA (SEQ ID NO: 79)

RASQPISISVH (SEQ ID NO: 80) FASQSIS (SEQ ID NO: 81) QQTFSLPYT (SEQ ID NO: 82) (see WO2008/76321).

In an embodiment, the anti-CD27 antibody or antigen-binding fragment thereof (eg, a humanized antibody) contains the CDRs below:

CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3

GFIIKATYMH (SEQ ID NO: 83) RIDPANGETKYDPKFQV (SEQ ID NO: 84)

YAWYFDV (SEQ ID NO: 85) RASENIYSFLA (SEQ ID NO: 86)

HAKTLAE (SEQ ID NO: 87) QHYYGSPLT (SEQ ID NO: 88) (see WO2012/04367).

Thus, the present invention includes compositions containing a PD1 and/or LAG3 binding molecule (eg, ISvD, such as a nanobody) (eg, F023700899; F023700931;

F023701017; F023700924; F023700969; F023700970;

F023701168; F023701173; F02370117 F023701176; 8;

F023701166; F023701171; F023701176; F023701162;

F023701172 or F023701177) associated with

F023701016;

F023701163;

F023701161;

F023701167;

pembrolizumab; and methods for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a PD1 and/or LAG3 binding molecule in combination with pembrolizumab (eg, pembrolizumab 200 mg administered once every three weeks). Optionally, another other therapeutic agent is also administered to the subject.

В варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is associated with an antibody pembrolizumab that contains an immunoglobulin heavy chain (or its CDR-H1, CDRH2 and CDR-H3) containing the amino acid sequence:

- 84 041987

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTN FNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK (SEQ ID NO: 89) ;

and an immunoglobulin light chain (or its CDR-Ll, CDR-L2 and CDR-L3) containing the amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLE

SEQ ID NO: 90

В варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is associated with an antibody containing an immunoglobulin heavy chain (or its CDR-H1, CDR-H2 and CDR-H3) containing the amino acid sequence:

QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRY

YADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 91);

and an immunoglobulin light chain (or its CDR-L1, CDR-L2 and CDR-L3) containing the amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP IARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDE

QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92).

В одном варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161 ; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is linked to any one or more of 13-cis-retinoic acid, 3-[5-(methylsulfonylpiperadimethyl)-indolyl]-quinolone, 5-hydroxy-phenolone, 4-hydroxy-phenolone, 5-deoxy-phenone, 5'-deoxy-5-fluorouridine, 5-fluorouracil, b-mecaptopurine, 7-hydroxystaurosporine, A-443654, abirateroanacetate, abraxane, ABT-578, acolbifene, ADS-100380, aflibercept, ALT-110, altretamine, amifostine, aminoglutethimide, amrubicine , amsacrine, anagrelide, anastrozole, angiostatin, AR-23573, ARQ-197, arzoxifene, AS-252424, AS-605240, asparaginase, ATI3387, AT-9263, atracentan, axitinib, AZD1152, Bacillus Calmette-Guérin (BCG) vaccines, batabulina, VS-210, without zodutox, bevacizumab, BGJ398, bicalutamide, Bioy, BIO140, VKM120, bleomycin, BMS-214662, BMS-247550, BMS-275291, BMS-310705, bortezimibe, buserelin, busulfan, calcitriol, camptothecin, canertinib, carsticitabine, carbine, mu CC8490, CEA (recombinant vaccine vaccinia-carcinoembional antigen), cediranib, CG-1521, CG-781, chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine, clodronate, cobimetnib, COL-3, CP-724714, cyclophosphamide cyproterone, cyproterone acetate, cytarabine, cypropyridine, cyprocytosine arabinoside, dabrafenib, dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib, daunorubicin, decatanib, degelin, denileukin, deoxycoformycin, depsipeptide, diarylpropionitrile, diethyl

- 85 041987 diftitox, DNE03, docetaxel, dovitinib, doxorubicin, droloxifene, endothecarin, yttrium-90 labeled edotreotide, edotreotide, EKB-569, EMD121974, encorafenib, endostatin, enzalutamide, enzastaurin, epirubicin, epitilone B, ERA9erbitux2, эрлотиниба, эстрадиола, эстрамустина, этопозида, эверолимуса, экземестана, фиклатузумаба, финастерида, флавопиридола, флуксуридина, флударабина, флудрокортизона, флуоксиэстерона, флутамида, схемы FOLFOX, фулвестранта, галетерона, ганетеспиба, гефитиниба, гемцитабина, гамицина, глюкопиранозиллипида А, гозерелина, гозерелинацетата, gossypol, GSK461364, GSK690693, HMR-3339, hydroxyprogesterone caproate, hydroxyurea, IC87114, idarubicin, idoxifen ifosfamide, IM862, imatinib, IMC-1C11, imiquimod, INC280, INCB24360, INO1001, interleukin-umabarino-2 , JNJ16241199, ketoconazole, KRX-0402, lapatinib, lasofoxifene, LEE011, letrozole, leucovorin, leuprolide, leuprolide acetate, le вамизола, липосомного паклитаксела, ломустина, лонафарниба, люкантона, LY292223, LY292696, LY293646, LY293684, LY294002, LY317615, LY3009120, маримастата, мехлорэтамина, медроксипрогестеронацетата, мегестролацетата, MEK162, мелфалана, меркаптопурина, месны, метотрексата, митрамицина, митомицина, митотана, митоксантрона , suspensions of thermally inactivated Mycobacterium obuense, tosasertib, MLN8054, natitoclax, neovastat, neratinib, neuradiab, nilotinib, niluthimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, otatumumab, oregovomab, ornatuzumab, orterol, oxalibonatumab, paclitaxel , pazopanib, PD0325901, PD184352, PEG-interferon, pemetrexed, pentostatin, perifosine, phenylalanine mustard, PI-103, pitilylib, PIK-75, pipenoxifene, PKI-166, plicamycin, poly-ICLC, porfimer, prednisone, procarbazine, progestins, polysaccharide , associated with the PSK protein (derived from Basidiomycete coriolus versicolor), PLX8394, PX-866, R-763, p алоксифена, ралтитрекса, разоксина, ридафоролимуса, ритуксимаба ромадипсина, RTA744, рубитекана, скриптейда, SdxlO2, селициклиба, селуметиниба, семаксаниба, SF1126, сиролимуса, SN36093, сорафениба, спиронолактона, скваламина, SR13668, стрептозоцина, SU6668, субероиланилалид-гидроксамовой кислоты, сунитиниба, synthetic estrogen, talampanel, talimogen-lagerparepvec, tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifen, testosterone, tetraandrin, TGX-221, thalidomide, b-thioguanine, thiotepa, ticilimumab, tifofarnib, tivozanib, TKI-258, TLK286, TNFa (factor tumor necrosis alpha), topotecan, toremifenecitrate, trabectedin, trametinib, trastuzumab, tretinoin, trichostatin A, triciribine phosphate monohydrate, triforelin pamoate, TSE-424, uramustine, valproic acid, valrubicin, vandetanib, vatalanib, VEGF traps, vemurafenib, vinblastine, vindesine, vindesine, vindesine , vinorelbine, vitaksin, vorinostat, VX-745, wortmanin, Xr311, Z-100, Bacillus tubercul extract osis in hot water, zanolimumab, ZK186619, ZK-304709, ZM336372, or ZSTK474.

В одном варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161 ; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is associated with one or more antiemetics, including but not limited to: casopitant (GlaxoSmithKline), netupitant (MGI-Hantelsinn receptor antagonists) (sold as Aloxy by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (sold as Benadryl® by Pfizer, New York, NY), hydroxyzine (sold as Atarax® by Pfizer, New York, NY), metoclopramide (sold as Raglan® by AH Robins Co, Richmond, VA), lorazepam (sold as Ativan® by Wyeth, Madison, NJ), alprazolam (sold as Xanax® by Pfizer, New York, NY), haloperidol (p born as Haldol® by Ortho-McNeil; Raritan, NJ), droperidol (Inapsin®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc, Marietta, GA), dexamethasone (sold as Decadron® by Merck and Co, Rahway, NJ), methylprednisolone (sold as Medrol® from Pfizer, New York, NY), prochlorperazine (sold as Compazine® from Glaxosmithkline, Research Triangle Park, NC), granisetron (sold as Kytril® from Hoffmann-La Roche Inc, Nutley, NJ), ondansetron (sold as Zofran® from Glaxosmithkline; Research Triangle Park, NC), dolasetron (sold as Anzemet® from Sanofi-Aventis, New York, NY), tropisetron (sold as Navoban® from Novartis, East Hanover, NJ).

Other side effects of cancer treatment include depletion of red and white blood cells. Соответственно, в варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F023701168; F023701173; F023701168 ; F023701176; 8; F023701161; F023701166, F023701171, F023701176, F023701162, F023701167, F023701172 или F023701177) связана с агентом, который лечит или предупреждает такое истощение, как, например, филграстим, ПЭГ-филграстим, эритропоэтин, эпоэтин-альфа или дарбепоэтин- alpha.

В варианте осуществления изобретения связывающая PD1 и/или LAG3 молекула (например, ISVD, такое как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177), associated with

- 86 041987 vaccine. In an embodiment of the invention, the vaccine is a cancer vaccine, a peptide vaccine, or a DNA vaccine. For example, in an embodiment of the invention, the vaccine is a tumor cell (eg, an irradiated tumor cell) or a dendritic cell (eg, a tumor peptide enriched dendritic cell).

В варианте осуществления изобретения связывающую PD1 и/или LAG3 молекулу (например, ISVD, такой как нанотело) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) are administered together with a therapeutic procedure. A therapeutic procedure is one or more steps performed by a physician or clinician in the treatment of a subject that are intended to alleviate one or more symptoms (e.g., cancer and/or infectious disease) in the subject being treated, either by reducing or eliminating such symptoms, or by inhibiting the development of such symptoms, for example a symptom of a cancer, such as tumor growth or metastasis, to any clinically measurable degree.

In an embodiment of the invention, the therapeutic procedure is anti-cancer radiation therapy. For example, in an embodiment of the invention, the radiation therapy is external beam radiation therapy (EBT): a method of delivering a high energy x-ray beam to a tumor site. The beam is generated outside the patient (for example, by a linear accelerator) and directed to the tumor site. These x-rays can destroy cancer cells, and careful treatment planning avoids damaging surrounding healthy tissue. No radioactive sources are placed inside the patient's body. In an embodiment of the invention, the radiation therapy is proton beam therapy: a type of conformal therapy that bombards diseased tissue with protons instead of x-rays. In an embodiment of the invention, the radiation therapy is conformal external beam radiation therapy: a procedure that uses advanced technology to tailor radiation therapy to the structures of the human body.

In an embodiment of the invention, radiation therapy is brachytherapy: the temporary placement of radioactive materials within the body, usually used to provide an additional dose or to enhance radiation in a specific area.

В варианте осуществления изобретения хирургической процедурой, проводимой совместно с введением связывающей PD1 и/или LAG3 молекулы (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) is the surgical removal of the tumor.

Therapeutic Options

The invention includes a method for preventing and/or treating at least one disease or disorder that can be prevented or treated with a PD1 and/or LAG3 binding molecule (e.g., an ISVD such as a nanobody) (e.g., F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176, 8, F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172, или F023701177) по настоящему изобретению, необязательно, в сочетании с дополнительным терапевтическим агентом или терапевтическим a method that includes administering to a subject in need thereof a pharmaceutically active amount of PD1 and/or LAG3 binding molecules and/or a pharmaceutical composition containing them.

Лечение или воздействие означает введение связывающих PD1 и/или LAG3 молекул (например, ISVD, таких как нанотело) по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176, 8, F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177) to a subject having one or more symptoms of a disease for which PD1 and/or LAG3 binding molecules are effective, e.g. disease, or suspected of having cancer or an infectious disease, against which the agent has therapeutic activity. Typically, PD1 and/or LAG3 binding molecules are administered in an effective amount or dose that will reduce one or more symptoms (e.g., cancer or infectious disease) in a treated subject or population, either by reducing or eliminating such symptoms, or by inhibiting the development of such symptoms, for example cancer symptoms such as tumor growth or metastasis, to any clinically measurable extent. The effective amount of the PD1 and/or LAG3 binding molecule may vary depending on factors such as the stage of the disease, the age and weight of the patient, and the ability of the drug to elicit the desired response in the subject.

The animal to be treated may be any warm-blooded animal, but in particular it may be a mammal and in particular a human. As will be understood by one of skill in the art, the subject to be treated will specifically be a human suffering from or at risk for the diseases and pathologies set forth herein. In general, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by the attending physician.

The invention also relates to a pharmaceutical composition that contains at least one amino acid sequence that binds a PD1 and/or LAG3 molecule (for example, ISVD, such as nanobody), polypeptide or compound described herein, such as F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177, and optionally at least one pharmaceutically acceptable carrier, diluent or excipient. Such preparations, carriers, excipients and diluents may generally be as described in Ablynx NV published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009 /068627.

Для получения фармацевтических или стерильных композиций связывающих PD1 и/или LAG3 молекул (например, ISVD, таких как нанотела) по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176 ; 8; F023701161, F023701166, F023701171, F023701176, F023701162, F023701167, F023701172 or F023701177), PD1 and/or LAG3 binding molecules are mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). Such compositions are part of the present invention.

В объем настоящего изобретения входят обезвоженные, например, лиофилизированные композиции, содержащие связывающие PD1 и/или LAG3 молекулы (например, ISVD, такие как нанотела) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701172 or F023701172 or F023701172 or F023701177) or a pharmaceutical composition thereof which does not substantially contain a pharmaceutical composition

Formulations of therapeutic and diagnostic agents can be prepared by mixing with suitable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) ) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

In general, for the prevention and/or treatment of the diseases and disorders set forth herein, and depending on the particular disease or disorder being treated, the efficacy and/or half-life of the specific fusion proteins or constructs to be used, and the route of administration used and the particular pharmaceutical formulation or composition, the nanoparticles and polypeptides of the invention are typically administered in an amount of 1 to 0.01 µg/kg body weight per day, preferably 0.1 to 0.1 µg per kg body weight per day, such as about 1 , 10, 100, or 1000 mcg/kg of body weight per day, either continuously (eg, by infusion), or as a single daily dose, or as several divided doses throughout the day. In General, the attending physician can determine the appropriate daily dose depending on the factors indicated herein. It will also be clear that in particular cases the attending physician may choose to deviate from these amounts, for example, based on the above factors and his expert judgment. In general, some administration amount recommendations can be derived from the amounts typically administered for comparable conventional antibodies or antibody fragments against the same target, administered in essentially the same route, but given differences in affinity/avidity, potency, biodistribution, half-life, and similar factors well known to the person skilled in the art.

Способ введения субъекту связывающих PD1 и/или LAG3 молекул (например, ISVD, таких как нанотела) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161, F023701166 , F023701171, F023701176, F023701162, F023701167, F023701172 or F023701177) may vary. Routes of administration include oral, rectal, mucosal, enteric, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intranasal, inhalation, insufflation, topical, dermal, transdermal, or intra-arterial.

The determination of the appropriate dose is made by the attending physician, for example, using parameters or factors known or expected in the art to influence treatment.

- 88 041987 As a rule, when determining the dosage, it is started with an amount slightly less than the optimal dose, and then gradually increased until the desired or optimal effect is achieved in comparison with any negative side effects. Important diagnostic parameters include symptoms such as inflammation or the level of inflammatory cytokines produced. In general, it is desirable that the biological compounds to be used be from the same species as the animal to be treated, thereby minimizing any immune response to the reagent. In the case of a human, for example, chimeric, humanized and fully human antibodies may be desirable. Recommendations for selecting appropriate doses of PD1 and/or LAG3 binding molecules (eg F023700924 or F023700931) are available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) ( 1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl J Med 348:601-608 Milgrom et al (1999) New Engl J Med 341:1966-1973 Slamon et al (2001) New Engl J Med 344:783- 792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

Whether a symptom of a disease has been relieved can be assessed using any of the clinical tests commonly used by physicians or other healthcare professionals to assess the severity or developmental status of that symptom. While an embodiment of the present invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptom(s) of the target disease in every subject, it should alleviate the symptom(s) of the target disease in a statistically significant number of subjects as determined by any statistical test known to in the field, such as Student's t-test, chi-square test, Mann and Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test.

Поскольку связывающие PD1 и/или LAG3 молекулы по настоящему изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167 ; F023701172 or F023701177) are capable of binding to PD1 (e.g. ISVD, e.g. nanobodies), they can in particular be used for the treatment or prevention of cancer, metastatic cancer, solid tumor, hematological cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, transitional cell bladder cancer, bladder cancer, Wilm's tumor, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer, non-small cell lung cancer, stomach cancer, colorectal cancer, cervical cancer uterus, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cellular carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic glioma, oligodenimomas choroid plexus, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid or liver cancer, breast cancer, and gastric cancer.

The PD1 and/or LAG3 binding molecules (eg, ISVDs such as nanobodies) of the present invention can be used to treat or prevent infectious diseases such as, for example, a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. In an embodiment of the invention, the viral infection is an infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), ebola virus, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV- 6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, virus vaccinia virus, HTLV virus, dengue fever virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus. In an embodiment of the invention, the bacterial infection is an infection with bacteria selected from the group consisting of chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Seracia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli , Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis and Borriella. In an embodiment of the invention, the fungal infection is an infection with fungi selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum. In an embodiment of the invention, the parasitic infection is an infection with a parasite selected from the group consisting of Entamoeba histo

- 89 041987 lytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

The present invention also includes methods for:

inhibition of LAG3 binding to MHC class II molecules;

competition with MHC class II molecules for binding to LAG3;

binding a LAG3 binding molecule, eg a PD1/LAG3 binding molecule, to native LAG3 on the surface of activated CD4+ and/or CD8+ T cells;

inhibition of LAG3 homodimerization; и/или стимулирования антигенспецифичной продукции IL-2 Т-клетками в организме субъекта путем введения субъекту связывающей PD1/LAG3 молекулы (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700924; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176 ; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177); or in vitro by contacting LAG3 with PD1/LAG3 binding molecules. This action may be mediated by LAG3 binding molecules. Thus, such methods can also be performed using any binding molecule that includes a LAG3 binding moiety.

The present invention also includes methods for:

blocking binding between PD1 and PD-L1 and/or PD-L2;

binding a PD1 binding, eg, PD1/LAG3 molecule to B cells and/or T cells;

blocking PD1-mediated T cell inhibition, T cell apoptosis, and/or T cell depletion in a subject by administering PD1/LAG3 binding molecules (eg, F023700924 or F023700931) to the subject; or in vitro by contacting PD1 with PD1/LAG3 binding molecules. Such actions may be mediated by a PD1 binding molecule. Thus, such methods can also be performed with any binding molecules that include a PD1 binding moiety.

The present invention further includes methods for increasing the half-life of a binding molecule, such as a PD1 and/or LAG3 binding molecule, by fusing the binding molecule to a human serum albumin binding moiety, such as ALB11002.

The invention also relates to methods for treating the above diseases and disorders, which typically include administering to a subject in need thereof (i.e. suffering from one of the above diseases) a therapeutically effective amount of PD1 and/or LAG3 binding molecules (e.g. ISVDs such as nanobodies ) по изобретению (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 или F023701177). The invention also relates to a PD1 and/or LAG3 binding molecule of the invention for use in the prevention or treatment of one of the aforementioned diseases or disorders.

Связывающие PD1 и/или LAG3 молекулы (например, ISVD, такие как нанотела) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; F023701172 or F023701177), the polypeptides, compounds and polynucleotides (eg vectors) described herein are preferably administered into the bloodstream. As such, they may be administered by any suitable route that allows PD1 and/or LAG3 binding molecules, polypeptides, compounds, and polynucleotides to be introduced into the blood stream, for example intravenously, by injection or infusion, or by any other suitable route (including oral, subcutaneous, intramuscular administration, administration through the skin, intranasal administration, administration through the lungs, etc.), which allows you to enter into the bloodstream PD1 and/or LAG3 binding molecules, polypeptides, compounds and polynucleotides. Suitable modes and routes of administration will be apparent to those skilled in the art, again, for example, from an examination of Ablynx NV published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or W02009/068627.

Настоящее изобретение также относится к устройству для инъекций, содержащему любые из связывающих PD1 и/или LAG3 молекул (например, ISVD, например, нанотел) (например, F023700899; F023700931; F023701016; F023701017; F023700924; F023700969; F023700970; F023701163; F023701168; F023701173 ; F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176; F023701162; F023701167; An injection device is a device that injects a substance into a patient's body via a parenteral route, such as intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector) that, for example, includes a barrel or container to hold the fluid to be injected (e.g., containing PD1 and/or LAG3 binding molecules or a pharmaceutical composition thereof).

- 90 041987 tsyu), a needle for piercing the skin and/or blood vessels for injection of liquid; and a plunger for pushing fluid out of the barrel and through the needle hole. In an embodiment of the invention, the injection device, which contains PD1 and/or LAG3 binding molecules or a pharmaceutical composition thereof) is an intravenous (IV) injection device. Such a device includes PD1 and/or LAG3 binding molecules or a pharmaceutical composition thereof) in a cannula or trocar/needle that can be attached to a hose that can be attached to a bag or reservoir to hold fluid (e.g. saline or lactate solution). Ringer containing NaCl, sodium lactate, KCl, CaCl2 and optionally including glucose) administered to the subject through a cannula or trocar/needle. The PD1 and/or LAG3 binding molecules or pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device after the trocar and cannula are inserted into the subject's vein and the trocar is removed from the inserted cannula. The HU device, for example, may be inserted into a peripheral vein (eg, in the hand or arm); superior vena cava or inferior vena cava or into the right atrium of the heart (eg, central intravenous catheter); or into the subclavian, internal jugular, or femoral vein and, for example, progress towards the heart until reaching the superior vena cava or right atrium (eg, central venous line). In an embodiment of the invention, the injection device is an auto-injector, a jet injector, or an external infusion pump. The jet injector uses a fine jet of high pressure liquid that penetrates the epidermis to introduce PD1 and/or LAG3 binding molecules or their pharmaceutical composition into the patient's body. External infusion pumps are medical devices that deliver PD1 and/or LAG3 binding molecules or their pharmaceutical composition to a patient in a predetermined amount. External infusion pumps may be electrically powered or may be mechanical. Different pumps operate on different principles, for example, a syringe pump holds fluid in the syringe reservoir, while a movable piston controls fluid delivery; an elastomeric pump holds the liquid in an expandable balloon reservoir, and pressure from the elastic walls of the balloon delivers the fluid. In a peristaltic pump, a set of rollers clamps a segment on a flexible tube, pushing the liquid forward. In a multichannel pump, fluids can come from multiple reservoirs at multiple speeds.

It should also be noted that the drawings, any sequence listing and experimental portion/examples are provided only to further illustrate the invention and should not be construed or taken as limiting the scope of the invention and/or the appended claims in any way, unless expressly stated otherwise.

Other aspects, embodiments, advantages and uses of the invention will become apparent from the further description herein.

Examples

These examples are intended to illustrate the present invention without being a limitation. The compositions and methods presented in the examples are part of the present invention.

Example 1 Binding of monovalent human anti-PD-l nanobody to CHO.Hpd-1.

Binding to cell-expressed human PD-1 was evaluated on CHO cells overexpressing human PD-1. Serial dilutions of the nanobodies were prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1x105 cells/well were transferred to a 96-well V-shaped plate and resuspended in 100 μl of diluted nanobodies. After a 30 minute incubation at 4°C, cells were washed with 100 µl/well assay buffer and resuspended in 100 µl/well 1 µg/ml anti-FLAG (Sigma, F1804) or anti-HIS (AbD Serotec, MCA 1396) solution antibodies. Samples were incubated for 30 minutes at 4°C, washed with 100 µl/well assay buffer and resuspended in 100 µl/well 5 µg/ml PE-labelled goat anti-mouse IgG solution (Jackson ImmunoResearch, 115-116-071) . Samples were incubated for 30 min at 4° C., washed and resuspended in 100 μl/well of 5 nM TOPRO3 solution (Life Technologies, T3606) prior to analysis for FACS CANTO II (BD). The data from these experiments are shown in Fig. 5.

This example demonstrated that the anti-human PD-1 monovalent nanobody F023700706 binds to human PD-1 similarly to the monovalent F023700275 nanobody from which it was derived.

Example 2 Binding of monovalent human anti-LAG-3 nanobody to 3A9.hLAG-3.

Binding to cell-expressed human LAG-3 was evaluated on 3A9 cells overexpressing human LAG-3. Serial dilutions of the nanobodies were prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1x105 cells/well were transferred to a 96-well V-shaped plate and resuspended in 100 μl of diluted nanobodies. After a 30 minute incubation at 4° C., cells were washed with assay buffer at 100 μl/well and resuspended in 100 μl/well of 1 μg/ml anti-FLAG (Sigma, F1804) antibody solution. Samples were incubated for 30 minutes at 4° C., washed with 100 μl/well assay buffer, and resuspended in 100 μl/well of a 5 μg/ml solution of PE-labelled goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples were incubated for 30 min at 4°C, washed and resuspended in 100 µl/well of 5 nM TO solution

- 91 041987

PRO3 (LifeTechnologies, TZbOb) before analysis on FACS CANTO II (BD). The data from these experiments are shown in Fig. 6.

This example demonstrated that the sequence-optimized monovalent anti-human LAG-3 nanobody F023700842 binds to human LAG-3 similarly to the original monovalent nanobody F02376611B09 from which it was derived.

Example 3 Binding of a multispecific nanobody containing an anti-human PD-1 nanobody to CHO.hPD-1 and 3A9.rhesusPD-1.

Binding to cell-expressed human PD-1 and rhesus monkey PD-1 was assessed in CHO cells overexpressing human PD-1 and 3A9 cells overexpressing macaque PD-1, respectively. Serial dilutions of the nanobodies were prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1x105 cells/well were transferred to a 96-well V-shaped plate and resuspended in 100 μl of diluted nanobodies. After a 30-minute incubation at 4°C, the cells were washed with 100 µl/well assay buffer and resuspended in 100 µl/well of a 3 µg/ml solution of ABH0074, a monoclonal antibody that recognized the nanobody binding fragment of albumin that increased the half-life. Samples were incubated for 30 minutes at 4°C, washed with assay buffer at 100 µl/well, and resuspended in 100 µl/well of a solution of 5 µg/ml PE-labeled goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071) . Samples were incubated for 30 min at 4° C., washed and resuspended in 100 μl/well of 5 nM TOPRO3 solution (Life Technologies, T3606) prior to analysis for FACS CANTO II (BD). The data from these experiments are shown in Fig. 7 (A-B).

This example demonstrated that the bispecific anti-human PD-1/LAG-3 nanobodies F023700931 and F023700924 bind to both human PD-1 and rhesus monkey PD-1. Data are also provided for monospecific, bivalent control nanobodies F023700933 (human anti-PD-1) and F023700962 (human anti-LAG-3).

Example 4 Binding of a multispecific nanobody containing an anti-human LAG-3 nanobody to CHO.hLAG3 and CHO.rhesus/cynoLAG3.

Binding to cell-expressed human and simian LAG-3 (the extracellular domain is identical in cynomolgus and rhesus monkeys, so the gene would refer to cynomolgus and rhesus monkey LAG-3) was evaluated in CHO cells overexpressing human LAG-3, as well as cynomolgus monkey and rhesus monkey. Serial dilutions of nanobodies were prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1x105 cells/well were transferred to a 96-well V-shaped plate and resuspended in 100 μl of diluted nanobodies. After a 30-minute incubation at 4°C, the cells were washed with 100 µl/well assay buffer and resuspended in 100 µl/well of a 1-3 µg/ml solution of ABH0074, a monoclonal antibody that recognized a nanobody that binds an albumin fragment that increases half-life. Samples were incubated for 30 min at 4°C, washed with 100 μl/well assay buffer, and resuspended in 100 μl/well of a 5 μg/ml solution of PE labeled goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples were incubated for 30 min at 4° C., washed and resuspended in 100 μl/well of 5 nM TOPRO3 solution (Life Technologies, T3606) prior to FACSArray (BD) analysis. The data from these experiments are shown in Fig. 8 (A-B).

This example demonstrated that the bispecific anti-human PD-1/LAG-3 nanobodies F023700931 and F023700924 bind to both human LAG-3 and rhesus monkey/cynomolgus monkey LAG-3. Data are also shown for monospecific, bivalent control nanobodies F023700933 (human anti-PD-1) and F023700962 (human anti-LAG-3).

Example 5 Biophysical analysis of PD-L1-Fc and PD-L2-FC blockade.

Ligand-competition assays were performed on CHO cells overexpressing human PD-1. Serial dilutions of the nanobodies were prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. Five-fold serial dilutions of nanobodies were prepared starting at 1 μM (2x) and pre-diluted in 11 nM (2x) PD-L1_human-hFc or 29 nM (2x) PD-L2_human-hFc in a total volume of 220 μl. Cells at 2x10 ⁴ /well were seeded in 96-well V-shaped plates and resuspended in 200 μl/well of diluted nanobody/ligand solutions. After incubation for 90 minutes at 4° C., cells were washed with assay buffer at 100 μl/well and resuspended in PE-labeled goat anti-human IgG (Southern Biotech, 2043-09). Samples were incubated for 30 minutes at 4°C, washed and resuspended in 100 μl/well of 5 nM TOPRO3 solution (Life Technologies, T3606) prior to analysis for FACS CANTO II (BD). The data from these experiments are shown in Fig. 9 (A-H).

This example demonstrates that a monovalent module (nanobody) to human PD-1, F023700706, binds to human PD-1, completely blocking its interaction with PD-L1 (Fig. 9(A)) and with PD-L2 (Fig. 9(B)), similar to the original monovalent module F023700275 from which it was derived. In addition, this example showed that the anti-human PD-1/LAG-3 bispecific nanobodies, F023700931 and F023700924, bound to human PD-1 and completely blocked its interaction with

- 92 041987

PD-L1 (Fig. 9(C)) and with PD-L2 (Fig. 9(D)). Data are also shown for sequence-optimized, monospecific, bivalent control nanobodies against human PD-1 (F023700933) and human LAG-3 (F023700962) (FIG. 9(E-F)). Finally, in this example, additional amino acid variants of the anti-human PD-1 monovalent module (nanobody) F023700929 were demonstrated to bind to human PD-1, completely blocking its interaction with PD-L1 (Fig. 9(G)) and PD-L2. (Fig. 9 (H)). It should be noted that F023700706 and F023700929 contained an identical anti-human PD-1 monovalent nanobody, either as a FLAG3-HIS6 fusion protein or as a HIS6 fusion protein, respectively.

Example 6 Biophysical analysis of LAG-3-Fc blockade.

Ligand-competition assays were performed on human Daudi cells, which have high endogenous expression of the major histocompatibility complex (MHC) class II. A dilution series of nanobodies was prepared in assay buffer: HBSS/2% FBS. Before the experiment, Fc receptors on Daudi cells were blocked with a human Fc blocking agent (BD Pharmingen, 564220) and 5 μg/ml goat IgG (Jackson ImmunoResearch, 005-000-003) for 30 minutes at 4°C. Five-fold serial dilutions of the nanobody were started at 2 μM (2x) and pre-diluted with 80 nM (2x) human LAG-3-hFc in a total volume of 120 μl, plated at 1x105 cells/well in 96-well V-shaped plates and resuspended in 100 μl/ well of nanobody/ligand dilutions. After a 30 minute incubation at 4°C, the cells were washed and resuspended in PE-labeled goat anti-human IgG (Southern Biotech, 204309). Samples were incubated for 15 minutes at 4° C., washed and resuspended in 100 μl/well of 5 nM TOPRO3 solution (Life Technologies, T3606) prior to analysis for FACS CANTO II (BD). The data from these experiments are shown in Fig. 10 (A-C).

This example demonstrated that the monovalent module (nanobody) F023700842 to human LAG-3 bound to human LAG-3, completely blocking its interaction with MHC class II, similar to the original monovalent nanobody F0237611B09 from which it was derived (Fig. 10 (A) ). In addition, in this example, it was demonstrated that the anti-human PD-1/LAG3 bispecific nanobodies, F023700931 and F023700924, bound to human LAG-3 and completely blocked its interaction with MHC class II (Fig. 10 (B-C)). Data are also provided for monospecific, bivalent control nanobodies F023700933 (human anti-PD-1) and F023700962 (human anti-LAG-3).

Example 7 Proximity dimerization analysis.

Proximity dimerization assay was used, which is based on a beta-galactosidase enzymatic activity assay system for complementary fragments. The assay uses a fusion protein produced with a tag called ProLink (PK) and a complementary protein in which an acceptor enzyme (EA) protein is fused to a second protein. U2OS cells were stably transfected with the extracellular domain of LAG-3 (1-477) fused to the EA subunit and the extracellular domain of PD-1 (1-199) fused to the PK subunit. The U2OS.LAG3(1-477)EA.PD1(1-199)-PK #9 cell line was seeded in quadruplicates at 5000 cells/well in DiscoverX CP5 seeding medium in 384-well plates. The cells were allowed to attach for 4 hours at 37° C. in a 5% carbon dioxide incubator. Then, 11 serial dilutions (1:3) of the nanobody were added to the cells and incubated overnight (16 hours) at 37° C. in a 5% carbon dioxide incubator. The PathHunter detection reagent was added to the wells, incubated for one hour at room temperature in the dark, and then the plate was read on an Envision luminometer. The data from these experiments are shown in Fig. 11 (A-B).

This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobody F023700931 simultaneously binds to human PD-1 and human LAG-3 expressed on the cell membrane as the PK and EA subunits combine to form the active enzyme, producing a light signal. (Fig. 11(A)). Separate monospecific bivalent control antibodies against human PD-1 (F023700933) and F023700962 against human LAG-3 could not simultaneously bind both targets and therefore did not give a light signal. The individual modules in the bispecific human anti-PD-1/LAG-3 nanobody, F023700931, were linked by a 35 amino acid glycine-serine linker (35GS). Bispecific human anti-PD-l/LAG-3 nanobodies, F023701016 and F023701017, contained identical anti-PD-l and anti-LAG-3 modules as F023700931, but the glycine-serine linker between these modules in the constructs was 20 amino acids long (20GS) or 9 amino acids long (9GS), respectively. This example showed that shortening the glycine-serine linker from 35GS to 9GS had minimal effect on the ability to simultaneously bind both targets on the cell membrane and generate a light signal. Taken together, these data indicate that the bispecific human anti-PD-1/LAG-3 nanobody F023700931 can bind both targets simultaneously if both targets are expressed on the same cell.

This example also demonstrated that the bispecific human anti-PD-l/LAG-3 nanobody F023700924 simultaneously binds to human PD-1 and human LAG-3 expressed on the cell membrane (FIG. 11(B)). The individual modules in the bispecific human anti-PD-1/LAG-3 nanobody, F023700924, are linked by a 35 amino acid glycine-serine linker (35GS). Bispecific human anti-PD-1/LAG-3 nanobodies, F023700969 and F023700970, contained identical modules

- 93 041987 anti-PD-l and aHTu-LAG-3 as F023700924, but the glycine-serine linker between these modules in the constructs was 20 amino acids long (20GS) or 9 amino acids long (9GS), respectively. This example showed that shortening the glycine-serine linker from 35GS to 9GS had minimal effect on the ability to simultaneously bind both targets on the cell membrane and generate a light signal. Taken together, these data indicated that the bispecific human anti-PD-l/LAG-3 nanobody F023700924 can bind both targets simultaneously if both targets are expressed on the same cell.

Example 8 Analysis using genetically modified Jurkat.hPD-1.IL21uc and THP1.PD-L1 cells.

Clone DT999A1 is a PD-1 transgene-expressing Jurkat cell clone with a luciferase reporter under IL-2 control (Jurkat.hPD-1.IL21uc). Jurkat.hPD-1.IL21uc cells were grown in RPMI medium (Corning Cellgro 10 040-CV) containing 10% temperature-inactivated FBS (Hyclone SH309W.03), 2 mM L-glutamine (Cellgro 25-005-CI), 2 μg /ml puromycin (Sigma P9620) and 0.5 mg/ml geneticin (Gibco 10131 027). Cells were subcultured twice a week after the initial cell seeding at a density of 2x105 cells/ml, and subcultured when the density exceeded 1x106 cells/ml. PD-L1 transgene-expressing THP-1 cells (THP-l.PD-Ll) were grown in RPMI medium containing 10% temperature-inactivated FBS, 2 mM L-glutamine, and 0.5 μg/ml puromycin. Cells were subcultured twice a week after initial seeding at a density of 3x105 cells/ml and subcultured when they reached a density of 1x106 cells/ml.

Biological analysis was performed using assay medium (RPMI medium without Phenol Red (Gibco 11835 030) supplemented with 10% dialyzed FBS (Hyclone, SH30079.03). A stock 600 μM human albumin solution (Sigma, A8763) was prepared. 120 μM) solution was prepared using assay medium and added 25 μl to cell culture treated white wall plates (Costar 3903). serial 10-fold dilutions of nanobodies 25 µl dilutions of nanobodies were added to albumin-containing plates with white walls treated for cell culture The solution of nanobodies was incubated with albumin for 20-30 min at room temperature THP-1.PD-L1 cells were collected from a vial T75 cells were centrifuged and resuspended in 10 ml of assay medium.The number of cells in suspension was counted and adjusted to obtain a cell suspension of 4x106 cells/ml. Jurkat.hPD-1.IL21uc cells were harvested from a T-75 flask, cells were centrifuged and resuspended in 10 ml of assay medium. The number of cells in the suspension was counted and adjusted to give a suspension of Jurkat.hPD-1.IL21uc cells at 1x106 cells/ml. An equal volume of THP-1.PD-L1 cells and Jurkat.hPD-1.IL21uc cells was mixed. To this suspension was added 2 ng/ml LPS (2x) and 100 ng/ml IFN-γ (2x). A 50 µl suspension containing stimulators [IFN-γ (R & D System 285-IF/CF)+LPS (Sigma L4391)] was added to the nanobody dilutions. Cells were incubated with nanobodies dilutions for approximately 22 hours in an incubator. After 22 hours, 10 μl of a 55 ng/ml anti-CD3 antibody solution (BD Pharmingen 555336, llx working solution) was carefully added and left in the incubator for another two hours. After two hours of incubation, a white film (Perkin Elmer 6005199) was glued to the bottom of the plate and 100 µl of One-Glo reagent (Promega E6120) was added.

Incubated with shaking at room temperature for 3 min. Luminescence was recorded on a tablet reader capable of recording luminescent radiation with an integration time of 0.1 s. Raw data in RLU (relative light units) can be plotted directly, or can be plotted in times of change in luciferase signal obtained by dividing the signal from the experimental wells by the signal from the wells that did not contain the nanobody. The data from these experiments are shown in Fig. 12 (A-E).

This example showed that the monovalent human anti-PD-l nanobody F023700706, by binding to human PD-l expressed by the T cell line, blocks the interaction of PD-l with PD-Ll expressed by the co-cultured cell line, reversing PD-Ll mediated suppression. T cell, thereby allowing the T cell to be more responsive to the T cell receptor agonist by inhibiting the PD-Ll mediated suppression process (FIG. 12(A)). In addition, this example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies, F023700931 and F023700924, by binding to human PD-l expressed by a T cell line, blocked the interaction of PD-l with PD-Ll, thereby allowing the T cell to be more responsive to the T cell receptor agonist by inhibiting the PD-Ll mediated suppression process (FIG. 12(B)). Data are also provided for monospecific bivalent control nanobodies against human PD-l (F023700933) and against human LAG-3 (F023700962). In addition, this example showed that the bispecific human anti-PD-1/LAG-3 nanobodies F023700969 and F023700970 containing 20GS or 9GS linkers, respectively, had similar activity to the parent F023700924 molecule containing the 35GS linker (Fig. 12 (C )). Similarly, bispecific human anti-PD-l/LAG-3 nanobodies F023701016 and F023701017 containing 20GS or 9GS linkers, respectively, had a similar

- 94 041987 activity with the original molecule F023700931 containing 35GS-linker (Fig. 12 (D)). In addition, this example demonstrated that additional amino acid variants of the sequence-optimized monovalent human anti-PD-1 nanobody F023700706 bind to human PD-1 expressed by a T cell line, blocking the interaction of PD-1 with PD-L1, thereby allowing the T cell to respond more to the T cell receptor agonist by inhibiting PD-L1-mediated T cell suppression (FIG. 12(E)).

Example 9 Assay using SEB-activated human PBMCs.

Human peripheral blood mononuclear cells (PBMC) were thawed in complete RPMI medium with 10% human serum (RPMI 1640 Glutamax (Thermo Fisher Scientific, cat. 1 11875085), 1x penicillin-streptomycin mixture (Thermo Fisher Scientific, cat. no. 15140148). ), lx beta-mercaptoethanol (Thermo Fisher Scientific, cat. no. 21985023), 1xck HEPES (Thermo Fisher Scientific, cat. no. 15630080), 1x sodium pyruvate (Thermo Fisher Scientific, cat. no. 11360070), 1x essential amino acids (Thermo Fisher Scientific, cat. no. 11360070), 11140076), human serum (Sigma, cat. 1 H4522100ML, Lot # SLBP2783V).Cells were resuspended to a density of 2.5x106 cells/ml and spaced 100 µl per well in a 96-well U-shaped plate at 2.5x105 cells/well A series of 3-fold dilutions was prepared to obtain 4-fold working solutions and a 6-point dose-response curve was prepared.50 μl of the nanobody solution was added to the wells and incubated for 15-30 minutes at room temperature, while preparing a race streptococcal endotoxin B (SEB) solution. The stock solution of SEB (Toxin Technology Inc, Cat. No. BT202) was prepared by reducing SEB to 1 mg/ml in distilled water and freezing it at -80°C. A 2 μM working solution was prepared by adding 2 μl per ml of medium, and the 2 μM working solution was serially diluted (1:10) to obtain a titration curve. Added 50 µl to the appropriate titration curve wells as an internal standard to confirm that the assay is proceeding as expected and to determine the maximum response that can be elicited from different donors. The working solution of SEB (2 μM) was diluted to a concentration of 40 nM and added 50 μl to the wells to stimulate PBMC (the final concentration of SEB was 10 nM). Incubated for 72 hours at 38°C and 5% CO ₂ . 100 ml of the supernatant was transferred to a new 96-well plate and diluted 1:2 for IL-2 analysis using the MSD V-plex human IL-2 kit (MesoScale Discovery, K151QQD-1). The residue was stored at -80°C. The data from these experiments are shown in Fig. 13 (AR).

This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 produced an increase in IL-2 levels after stimulation with multi-donor peripheral blood mononuclear cell (PBMC) T cell receptor (PBMC) agonists (A-R).

Example 10 Mixed Lymphocyte Population (MLR) Response Assay.

Human peripheral blood mononuclear cells (PBMCs) were purified from leukocytes and frozen in a liquid nitrogen cryostat. Frozen human PBMCs were thawed, diluted in complete RPMI (RPMI supplemented with 10% human serum), centrifuged at 450xg for 5 minutes, and the cell pellet resuspended in complete RPMI (Gibco RPMI 1640 medium (Thermo fisher Scientific, 11875-119) with human serum (Sigma-Aldrich, H4522-100 mL) Monocytes were enriched using a human monocyte enrichment kit (STEMCELL technologies, 19059) Cells were transferred into b-well plates at a density of 1x106 cells/mL (5 mL/well) in complete RPMI containing 100 ng/ml GM-CSF (R & D Systems, 215-GM-110) and 50 ng/ml human IL-4 (R & D Systems, 204-IL010/CF) Monocytes were incubated at 37° C for 5 days to obtain dendritic cells (DC) Monocyte-derived dendritic cells (Mo-DC) were harvested on day 6, counted and used in MLR assay as stimulators.

On the day of the start of the experiment, frozen human PBMCs were thawed and diluted twice in complete RPMI containing penicillin/streptomycin. CD4+ T cells from each donor were enriched using the EasySep Human CD4 T4 cell isolation kit (STEMCELL technologies, 17952). The isolated CD4 ⁺ T cells were resuspended at a density of 1x106 cells/ml in complete RPMI. Mo-DC was mixed at a 1:10 ratio (1x105 cells/ml) with CD4 ⁺ T cells (1x106 cells/ml) and the cell mixture was seeded in a 96-well U-shaped plate at 200 μl/well. The nanobodies were serially diluted (1:4) and 5-fold working solutions were obtained. 50 μl of each dilution was added to 200 μl of culture to give 1x the final concentration of nanobodies. Culture supernatants were collected 5 days after the start of the experiment for IFNy quantification using the V-plex Human Proinflammatory Panel I (Mesoscale Discovery, K15052D-1). The data from these experiments are shown in Fig. 14 (AF).

This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 increased IFN-gamma levels after primary stimulation of CD4+ T cells with allogeneic Mo-DCs obtained from different donors (A-F).

Example 11 Analysis using genetically modified 3A9.hLAG-3 cells.

The 3A9.hLAG3 line is a human murine T

- 95 041987 3A9 cell hybridoma. The LK35.2 lineage (ATCC; HB-98) is a murine B cell hybridoma that carries surface molecules I-Ad ^k and I-Ed ^k that present a specific antigen (namely, chicken egg lysozyme (HEL) peptide DGSTDYGILQINSRWW) limited MHC class II T-cell hybridoma 3A9. 3A9.hLAG3 cells were counted and resuspended at a density of 4x10 ⁶ cells/ml in fresh medium [RPMI (Invitrogen, 61870 036) supplemented with 10% FBS and penicillin/streptomycin]. LK35.2 cells were counted and resuspended at a density of 1x10 ⁶ cells/ml in fresh medium. 25 μl of 3A9.hLAG3 cells (1E5 cells) were added to the wells of a 96-well flat bottom plate (Corning 3610). Prepared 4x (200 μm) solution of human albumin (Sigma, A8763) and added 25 μl to the cells. The nanobodies were diluted to a concentration of 5x in complete medium, and 20 μl of a 5-fold solution of nanobodies was added to the cells. 3A9.hLAG3 cells were incubated with nanobodies for 30 min at 37°C. A 500 μM stock solution of HEL peptide was prepared. (GenScript custom peptide) and stored at 20°C. The HEL peptide stock solution was diluted 1:20 (up to 25 μM) in cell culture medium, and then the HEL peptide was further diluted 1:833 in LK35.2 cells (final HEL concentration=~30 nM). LK35.2 cells were incubated with the peptide for 30 minutes at 37°C. 33 μl of peptide-exposed LK35.2 cells were added to a 96-well plate containing nanobody-exposed 3A9.hLAG 3 cells. The culture was incubated at 37°C for 24 h. analysis for IL-2 (IL-2 Mesoscale V-Plex, Mesoscale K152QQD-4). The data from these experiments are shown in Fig. 15 (AD).

This example showed that the monovalent human anti-LAG-3 nanobody F023700842 binds to human LAG-3 expressed by a T cell line and blocks the interaction of LAG-3 with MHC class II expressed by a co-cultured cell line, reversing T cell suppression mediated by MHC class II, thereby allowing the T cell to respond more to the T cell receptor agonist due to inhibition of MHC class II mediated suppression. The class II MHC inhibition efficacy of monovalent human anti-LAG-3 nanobody F023700842 was similar to that of the parent aF023700656 from which it was derived (FIG. 15(A)). In addition, this example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 bind to human LAG-3 expressed by a T cell line and block the interaction of LAG-3 with MHC class II, thereby allowing T β-cell respond more to the T-cell receptor agonist due to the inhibition of class II MHC-mediated suppression (FIG. 15(B)). Data are also provided for monospecific bivalent control nanobodies against human PD-1 (F023700933) and against human LAG-3 (F023700962). In addition, this example showed that the human anti-PD-1/LAG-3 bispecific nanobodies F023700969 and F023700970 containing 20GS or 9GS linkers, respectively, had similar activity relative to the parent F023700924 molecule containing the 35GS linker (Fig. 15 (WITH)). Similarly, the bispecific human anti-PD-1/LAG-3 nanobodies F023701016 and F023701017 containing 20GS or 9GS linkers, respectively, had similar activity to the parent F023700931 molecule containing the 35GS linker (FIG. 15(D)).

Example 12. Biological analysis using genetically modified Jurkat.hPD-1.LAG-3 cells.

Creation of Jurkat.LAG-3.PD-1 cells (clone DT1088-G10PD1).

The human LAG-3 transgene was introduced into Jurkat (Je6.2.11) cells using a retroviral delivery system with puromycin as a selectable marker. To select a clone (DT1088G10) with optimal LAG-3 and CD3 expression, the limiting dilution method was used. The human PD-1 transgene was introduced into clone DT1088G10 using a lentiviral delivery system with genticin as a selectable marker. Cells were sorted on FACS (fluorescent activated cell sorter) for high levels of LAG-3 and PD-1 expression. A pool of sorted cells was maintained and subcultured in complete RPMI medium [RPMI (Corning 10-040-CV) supplemented with 10% FBS (Hyclone SH30910.03), 1 mM sodium pyruvate (BioWhittaker, 13-115E), 2 mM L-glutamine ( Corning CellGro, 25005-CI), 10 mM HEPES (Corning, 26-060-CI), 1x essential amino acids (Sigma, M7145), 0.2 μg/ml puromycin (Sigma P9620), and 0.5 mg/ml G418 ( Gibco 10131-027)].

Creation of expressing Raji.PD-L1 cells.

The human PD-L1 transgene was introduced into Raji cells (ATCC CCL-86) using a retroviral delivery system with puromycin as a selectable marker. Cells were sorted for enrichment for expression of PD-L1 and MHC class II (endogenous). The enriched cell pool was maintained and subcultured in complete RPMI medium (RPMI medium supplemented with 10% FBS (Hyclone), 2 mM L-glutamine, 10 mM HEPES, and 0.25 μg/ml puromycin).

The stock solution of SED-toxin was prepared by reducing SED (Toxin technology, DT303) to a concentration of 1 mg/ml in sterile distilled water and stored at -80°C. Raji.PD-L1 cells were preloaded with toxin by incubating a 0.27x106 Raji-PD-L1/mL cell suspension with SED toxin (1.3x=130 ng/mL, final concentration 100 ng/mL) in RPMI medium containing 10% dialyzed FBS (Hyclone SH30079.03), for 30 minutes at 37°C in an incubator. Prepared suspen

- 96 041987 cells Jurkat.LAG-3.PD-1 with a density of 8x10 ⁶ /ml in RPMI medium containing 10% dialyzed FBS. 8-point serial 4-fold dilutions of nanobodies (12x=120 μg/ml) were prepared with an initial concentration in the assay of 10 μg/ml.

µl of Jurkat.LAG-3.PD-1 cell suspension (8x10 ⁶ cells/ml) was incubated with 45 µl of diluted nanobodies at room temperature for 30 minutes in a round bottom plate (Corning 3359). After 30 minutes, 36 μl of human albumin (Sigma, A8763; 15x=450 μM) was added to the mixture of Jurkat.LAG-3.PD-1 cells and nanobody to obtain a final concentration in the assay of 30 μM. 115 µl of SED-loaded Raji.PD-L1 cells were added to assay plates (Thermo Scientific No. 167008). SED-loaded Raji.PD-Ll cells were carefully layered with 35 µl of a mixture of Jurkat.LAG-3.PD-1 cells, nanobodies, and albumin. The assay plates were incubated for 24 hours at 37° C. in a 5% CO ₂ incubator. After a 24 hour incubation, 75 ml of the supernatant was transferred to a plate (Corning, No. 3605) and frozen at -80°C. Samples were analyzed using a V-plex MS-IL-2 kit (Mesoscale devices, cat. no. K151QQD). EC ₅₀ values were calculated using GraphPad Prism software. The data from these experiments are shown in Fig. 16 (A-B).

This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 bind to both human PD-1 and human LAG 3 expressed by a T cell line, block the interaction of PD-1 with PD-L1 , block the interaction of LAG-3 with MHC class II, thereby allowing T cells to be more responsive to the T cell receptor agonist by inhibiting the dual suppression of T cells mediated by PD-L1 and MHC class II (Fig. 16(A) ). Monospecific bivalent control nanobodies against human PD-1 (F023700933) and against human LAG-3 (F023700862) were only able to block one of the two inhibitory mechanisms, resulting in a much more modest T cell response to the T cell receptor agonist. In addition, this example showed that the bispecific human anti-PD-l/LAG-3 nanobodies F023700969 and F023700970 containing 20GS or 9GS linkers, respectively, had similar activity to the original F023700924 molecule containing the 35GS linker. Similarly, the bispecific human anti-PD-1/LAG-3 nanobodies F023701016 and F023701017 containing 20GS or 9GS linkers, respectively, had similar activity to the parent F023700931 molecule containing the 35GS linker (FIG. 16(B)).

Example 13 Analysis using a human T cell clone and JY.hPD-L1.

Creation and cultivation of a clone of human CD4+ T cells MHC class II alloantigen-specific CD4+ T cell clone BC4-49 was created by 2 rounds of reaction of a mixed population of leukocytes with the EBV-transformed B cell line JY and cloned at limiting dilutions. The clone was restimulated with allospecific antigens at 2 week intervals and cultured in Issel's medium (IMDM, Gibco 12440-053, with human serum (AB), Gemimi 100512, penicillin/streptomycin, Mediatech 30-002-CI, human albumin, Sigma A9080, ITS-X, Gibco 51500056, transferrin, Roche 10652202001, PA Bioxtra Sigma p5585, LA-OA-albumin, Sigma L9655). Fresh PBMCs were isolated from two human buffy coats obtained from the Stanford Blood Center and pooled in a 1:1 cell ratio. Before use, the PBMCs were irradiated in a gamma emitter with a dose of 4000 rad. Wild-type JY cells were prepared and irradiated with a dose of 5000 rad. T cell clones were cultured with feeder cells in a 24-well plate in a volume of 1 ml per well with final concentrations of CD4+ T cells 0.2x10 ⁶ /ml, PBMC irradiated 1x10 ⁶ /ml, JY irradiated 0.1x10 ⁶ /ml and 100 ng/ ml PHA (Sigma L9017). Recombinant human IL-2 (R&D Systems, 202-IL/CF) was added to a final concentration of 100 ng/ml on day 3 post-restimulation and replenished every 3-4 days throughout the cell expansion process. The cells were passaged to an optimal concentration between 0.5-1.0x10 ⁶ /ml. On the 7th day after restimulation, LAG-3 was expressed at a high level and PD-1 at an average level on the surface of T cells.

Functional assay with human CD4+ T cells Aloloantigen-specific CD4+ T cells were harvested from 24-well culture plates on day 7 after antigen re-stimulation, then washed twice with 20 ml PBS (Hyclone, SH3002802) containing 2 mM EDTA (Invitrogen , 15575-38), using centrifugation. The pellets were resuspended to a single cell suspension in Issel's medium. For test nanobodies, 5-fold serial dilutions were prepared in Issell's medium, starting at the highest concentration of 133 nM for a total of 7 dilutions in a volume of 100 μl in 96-well U-shaped culture plates (Falcon, 353077). Fifty microliters of a T cell suspension at a density of 4x10 ⁵ cells/ml was added to the wells containing the nanobody dilutions. The mixture of nanobodies and T cells was preincubated for 1 hour in an incubator at 37°C with 5% CO2. The human PD-L1 transgene expressed by JY cells (JY.hPD-L1) was used in co-culture to provide allospecific antigens. JY.hPD-L1 cultured in T-75 flask (Thermo Scientific, 156499) in RPMI medium (Corning Cellgro, 10-040-CV) with 10% FCS were harvested and irradiated with a 5000 rad gamma irradiator, then washed twice PBS containing 2 mM EDTA by centrifugation. The pellet was resuspended in Issel's medium and filtered through a 40 µm cell filter before seeding. 50 µl/well of JY.hPD-L1 suspension with a density of 2x10 ⁵

- 97,041,987 cells/ml were added to the pre-incubated mixture of nanobodies and T cells, the ratio of T cells to JY.hPD-L1 cells was 2:1. All points were performed in duplicate. After approximately 3 days of culture, 100 μl of supernatant/well was collected for human IFNy quantitation. IFNy ELISA was performed to assess IFNy levels for a pool of supernatants from duplicates using the hIFNy Quantikine kit (R&D Systems, SIF50). The analysis was performed according to the standard protocol provided by the manufacturer. EC ₅₀ values were calculated using GraphPad Prism software. The data from these experiments are shown in Fig. 17(A-C).

This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 bound both human PD-1 and human LAG-3, an expressed T cell clone, blocking the interaction of PD-1 with PD- L1, blocked the interaction of LAG-3 with MHC Class II, thereby allowing T cells to be more responsive to allogeneic stimulation due to inhibition of the dual suppression mediated by PD-L1 and MHC class II (Fig. 17 (A)). Monospecific bivalent control nanobodies against human PD-1 (F023700933) and against human LAG-3 (F023700862) were able to block only one of the two inhibitory mechanisms, resulting in a much lower T cell response to allogeneic stimulation. In addition, this example showed that the bispecific human anti-PD-1/LAG-3 nanobodies F023700969 and F023700970 containing 20GS or 9GS linkers, respectively, had similar activity to the original F023700924 molecule containing the 35GS linker (Fig. 17 (WITH)). Similarly, the bispecific anti-human PD-1/LAG-3 nanobodies F023701016 and F023701017 containing 20GS or 9GS linkers, respectively, had similar activity to the parent F023700931 molecule containing the 35GS linker (FIG. 17(B)).

Example 14 Evaluation of the binding of pre-existing antibodies (pre-antibodies) to F023700924 and F023700931.

The binding of preantibodies to nanobodies captured on human serum albumin (HSA) was assessed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH 7.4, 0.005% Tween 20) was used as assay buffer and experiments were performed at 25°C. The ligand lanes of the ProteOn GLC Sensor Chip were activated with EDC/NHS (flow rate 30 μl/min), and human serum albumin was applied at 10 μg/ml in ProteOn Acetate pH 4.5 buffer (flow rate 100 μl/min) to obtain immobilization at approximately 3600 RU. After immobilization, the surfaces were deactivated with ethanolamine-HCl (flow rate 30 μl/min). The nanobodies were run for 2 minutes at 45 μl/min across the HSA surfaces to obtain a nanobody capture level of approximately 600 RU for trivalent F023700924 and approximately 1000 RU for pentavalent F023700931. Samples containing pre-existing antibodies were diluted 1:10 in PBS-Tween20 (0.005%) before a 2 minute run at 45 µl/min followed by a 400 second dissociation step. After each cycle (ie, before the new nanobody capture step and the blood run step), the HSA surfaces were regenerated with a 2 minute HCl (100 mM) run at 45 μl/min. Sensorgram data processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing binding by pre-existing antibodies were obtained after double normalization by subtracting 1) nanobody-HSA dissociation and 2) non-specific binding in a control lane containing only HSA. The levels of binding to preantibodies were determined at a given point of 125 s (5 s after the end of binding). As a control, samples containing pre-existing antibodies were also tested for binding to a trivalent nanobody that had not been modified to reduce binding to these pre-antibodies (T013700112). The data from these experiments are shown in Fig. 19(A-I).

The human PD-1/LAG-3 bispecific nanobody, F023700924, contained three modules (one module for PD-1, one module for LAG-3, and one module for albumin); while F023700931 is a five-module nanobody (duplicate modules on PD-1, duplicate modules on LAG-3, and one module on albumin). One component of the sequence optimization approach was the inclusion of amino acid substitutions in the nanobody framework sequences that reduced the reactivity present in human serum to the nanobody modules. This example demonstrated that the pre-existing reactivity found in a panel of sera from healthy donors to the 3-module bispecific anti-PD-1/LAG-3 human nanobody F023700924 was reduced compared to the pre-antibody reactivity to another 3-module nanobody T013700112, which did not contains these framework amino acid substitutions (at two time points). The reactivity for the 5-module bispecific human anti-PD-1/LAG-3 nanobody F023700931 was higher than for the 3-module nanobody F023700924 (Fig. 19 (A-B)). In addition, this example demonstrated that the preantibody reactivity found in a panel of sera from cancer patients to the 3-module bispecific human anti-PD-1/LAG-3 nanobody F023700924 was reduced compared to the preantibody reactivity to the other 3-module nanobody T013700112. which did not contain these amino acid substitutions in the framework region (at two times

- 98 041987 exchange points). The reactivity for the 5-module bispecific human aHTu-PD-1/LAG-3 nanobody F023700931 was higher than for the 3-module nanobody F023700924 (Fig. 19(C-D)). Finally, the reactivity of a panel of sera from cancer patients for F023700924 and F023700931 was separated based on the type of cancer (FIG. 19(E-I)).

Example 15 Affinity Measurement of Sequence-Optimized Nanobodies Against Human LAG-3 Using Surface Plasmon Resonance.

The kinetic analysis of nanobodies was performed using the surface plasmon resonance (SPR) method using a Biacore T100 instrument (GE Healthcare). HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol% Surfactant P20) was used as assay buffer and experiments were performed at 25°C. Two Series S Sensor Chip CM5 flow lanes were activated with EDC (200 mM)/NHS (50 mM), and human anti-IgG (Fc) (GE Healthcare, BR100839) was passed at 5 μg/mL in the supplied immobilization buffer (10 mM sodium acetate, pH 5.0) to obtain an immobilization level of approximately 2000 RU. After immobilization, the surfaces were deactivated with 1M ethanolamine/HCl (pH 8.5). The flow rate during sensor preparation was set to 5 µl/min.

In a kinetic assay, human LAG3-hFc (62.5 nM) was passed for 1 minute at 10 μl/min over a single surface of human anti-IgG (Fc) to obtain a capture level of approximately 600 RU. 2.5-fold serial dilutions of nanobodies (namely, 5 μM to 3.3 nM for F023700842) were prepared in assay buffer before running for 2 minutes at 45 μl/min followed by dissociation for 900 s. After each cycle (i.e., before a new nanobody run and human-hFc LAG3 capture), anti-hIgG ^^ surfaces were regenerated by a 2-minute MgCl ₂ (3M) sweep at a rate of 10 μl/min.

Sensorgram processing and data analysis were performed using Biacore T100 Evaluation Software Version 2.0.4 (GE Healthcare). Sensorograms showing nanobodies binding were obtained after double normalization by subtracting 1) dissociation of LAG3-human αhFc/anti-hIgG (Fc) and 2) non-specific binding to the reference flow channel. The processed curves were evaluated by fitting to the mass transfer Langmuir model.

This example demonstrated that the human LAG-3 monovalent nanobody F023700842 bound human LAG-3 with high affinity.

Table E

Measurement of the affinity of nanobodies to human LAG-3 using surface plasmon resonance

kd(l/a) KD(M) P023700ta Human PD-1-Fc 2.0EW5 5.EE-04 2.9E-S FO237O1192 2.5E+06 3.2E-O3 1, ZE-09 F023701193 3.2E+O6 7.5E-03 2.4E-09 F023700S42 IE-Z-Es "evig 9.2E+05 1.9E-02 2.1E-08 F023700924 Coming Soon^ 6.4E+04 7.7E-03 1.2E-07 FO237OO931 3.2E+04 S.2E-03 2.5E-07 F023700924 shkaka- r: ΐ 2 y 2 £ b.9E" 7.9E-03 1.2E-07 F023700931 12E+O4 S-4E-03 2.7ΕΌ7

Example 16 Affinity Measurement of Sequence-Optimized Nanobodies Against Human PD1 Using Surface Plasmon Resonance.

The kinetic analysis of nanobodies was performed by the SPR method using a Biacore T100 instrument (GE Healthcare). HBS-EP+ (O,O1 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol% Surfactant P20) was used as assay buffer and experiments were performed at 25°C. Two Series S Sensor Chip CM5 flow lanes were activated with EDC (200 mM)/NHS (50 mM). hPD-1-hFc was injected (4 times) at a concentration of 5 μg/ml in an appropriate immobilization buffer (10 mm sodium acetate, pH 4.5) to obtain an immobilization level of approximately 303 RU. After immobilization, the surfaces were deactivated with 1M ethanolamine/HCl (pH 8.5). The flow rate during sensor preparation was set to 5 µl/min.

In kinetic analysis, 2.5-fold serial dilutions of nanobodies (namely, from 1 μM to 0.46 nm for F023700929, F023701192 and F023701193) were prepared in assay buffer before administration for 2 minutes at 45 μl/min followed by dissociation for 900 s. After each cycle (ie, before the new nanobody step), the hPD1-hFc surfaces were regenerated by a 1 minute injection of 10 mM glycine, pH 1.5, at 45 μl/min. Sensorgram processing and data analysis were performed using Biacore T100 Evaluation Software Version 2.0.4 (GE Healthcare). Sensorograms showing nanobodies binding were obtained after double normalization by subtracting 1) non-specific binding to the reference flow channel and 2) HBS-EP+ buffer run data. The processed curves were evaluated by fitting to the mass transfer Langmuir model.

This example demonstrated that a monovalent anti-human PD-1 antibody with high affinity

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Claims (18)

VOM binds to human PD-1. In addition, this example demonstrated that additional amino acid variants of the optimized sequence of the monovalent anti-human anti-PD-1 nanobody F023700929 (namely, F023701192 and F023701193) bind with high affinity to human PD-1. Table F Measurement of the affinity of sequence-optimized nanobodies for human PD-1 using surface plasmon resonance hMl/HcJ 2.0EMK 2.5E*® 12Mb URmax) KD (Μ) YAYU 2.EE-10 kt(RU/Ms) 23&E IS ao ise-® as a 450 24E-® 42M7 12 4 Example 17 Measurement of the affinity of a sequence-optimized polyspecific nanobody for human PD-1/LAG-3 for albumin from various species using surface plasmon resonance. The kinetic analysis of nanobodies was performed by the SPR method using a Biacore T100 instrument (GE Healthcare). HBS-EP+(O,O1 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol% Surfactant P20) was used as assay buffer and experiments were performed at 25°C. Four Series S Sensor Chip CM5 flow lanes were activated with EDC (200 mM)/NHS (50 mM), and human serum albumin (Sigma, A3782, lot SLBD72 04V) or rhesus monkey serum albumin (BioWorld, cat. no. 22070099-1 , lot L15091001DA) was run at 5 μg/ml in 10 mM sodium acetate, pH 4.5 to obtain immobilization levels of 179-312 RU in three flow lanes, respectively. After immobilization, the surfaces were deactivated with 1M ethanolamine/HCl (pH 8.5). The speed during sensor preparation was set at 5 µl/min. In the kinetic assay, 3-fold serial dilutions of nanobodies (namely, 6 μM to 2.7 nM) were prepared in assay buffer before running for 2 min at 45 μl/min followed by dissociation for 900 s. After each cycle (that is, before the introduction of a new concentration of nanobodies), all surfaces were regenerated with a 10-second injection of 10 mM glycine, pH 1.5, at a rate of 100 μl/min. Sensorgram processing and data analysis were performed using Biacore T100 Evaluation Software Version 2.0.4 (GE Healthcare). Sensorgrams showing nanobodies binding were obtained after double normalization by subtracting 1) non-specific binding to the reference flow channel and 2) the mean of two runs of HBS-EP+ buffer on the working flow channels. The processed curves were evaluated by fitting to the Langmuir model with mass transfer. This example demonstrated that the bispecific human anti-PD-1/LAG-3 nanobodies F023700931 and F023700924 bind to both human and rhesus monkey albumin with similar affinity. Table G Measurement of the affinity of sequence-optimized polyspecific human anti-PD-1/LAG-3 nanobodies for albumin from various species by surface plasmon resonance CLAIM 1. LAG-3 binding molecule containing an immunoglobulin single variable domain (ISVD) that binds to LAG-3, containing CDR1 containing the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65) or DYVMG (amino acids 6-10 of SEQ ID NO: 65); CDR2 containing the amino acid sequence AISESGGRTHYADSVKG (SEQ ID NO: 66) or AISESGGRTH (amino acids 1-10 of SEQ ID NO: 66); CDR3 containing the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67). 2. The LAG-3 binding molecule according to claim 1, wherein the immunoglobulin single variable domain contains the amino acid sequence EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64); or the specified sequence containing the E1D mutation. 3. A bispecific binding molecule that binds to PD1 and LAG3, containing an immunoglobulin single variable domain (ISVD) that binds to PD1, containing - 100 041987 CDR1 containing the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2 containing the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4; where X is W or V) or VITXSGGITY (SEQ ID NO: 7; where X is W or V); And CDR3 containing the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5, where X is W or F); and a single immunoglobulin variable domain that binds to LAG-3 containing a CDR1 containing the amino acid sequence GRTFSDYVMG (SEQ ID NO: 65) or DYVMG (amino acids 6-10 of SEQ ID NO: 65); a CDR2 containing the amino acid sequence AISESGGRTHYADSVKG (SEQ ID NO: 66) or AISESGGRTH (amino acids 1-10 of SEQ ID NO: 66); and CDR3 containing the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67). 4. The bispecific binding molecule of claim 3, wherein the single immunoglobulin variable domain that binds to PD1 contains the amino acid sequence DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTL VTVSS (SEQ ID NO: 57); EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO: 99); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (amino acids 1-119 of SEQ ID NO: 101); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 103); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITY YADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 104); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITY YADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS (SEQ ID NO: 105); single immunoglobulin variable domain that binds to LAG-3 contains the amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREFVAAISESGGRTH YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSS (SEQ ID NO: 64); or DVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREFVAAISESGGRTH YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCATTLLWWTSEYAPIKANDYDYWGQGTL VTVSS (amino acids 1-128 of SEQ ID NO: 96). 5. A binding molecule according to any one of claims 1 to 4, further comprising a C-terminal extension, wherein the C-terminal extension is alanine. 6. Polyspecific binding molecule that binds to PD1 and LAG3, containing PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 99; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 99; - 101 041987 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 99; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 20GS linker containing the amino acid sequence shown in SEQ ID NO: 100; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 57; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105 (D1E); 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105 (D1E); 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; - 102 041987 LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 105; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104 (D1E); 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104 (D1E); 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 104; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (N73S); 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (D1E, N73S); 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (N73S); 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (D1E, N73S); 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; - 103 041987 HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (N73S); 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 35GS linker containing the amino acid sequence shown in SEQ ID NO: 58; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine or PD1-ISVD containing the amino acid sequence shown in SEQ ID NO: 103 (N73S); 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; LAG3-ISVD containing the amino acid sequence shown in SEQ ID NO: 64; 9GS linker containing the amino acid sequence shown in SEQ ID NO: 125; HSA-ISVD containing the amino acid sequence shown in SEQ ID NO: 59; and C-terminal alanine. 7. A polyspecific binding molecule according to claim 6, containing an amino acid sequence selected from SEQ ID NOs: 106-124. 8. A bispecific binding molecule according to any one of claims 3 or 4, further comprising a half-life increasing fragment, where the half-life increasing fragment is an ISVD to human serum albumin that binds to human serum albumin, containing CDR1 containing the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60) or SFGMS (amino acids 1-5 of SEQ ID NO: 60); CDR2 containing the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL (amino acids 1-10 of SEQ ID NO: 61); And CDR3 containing the amino acid sequence GGSLSR (SEQ ID NO: 62). 9. The bispecific binding molecule of claim 8, wherein the human serum albumin ISVD contains the amino acid sequence EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSA (SEQ ID NO: 59). 10. The bispecific binding molecule of claim 3 further comprising a half-life increasing moiety, wherein the half-life increasing moiety is an ISVD to human serum albumin that binds to human serum albumin, comprising CDR1 containing the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60) or SFGMS (amino acids 1-5 of SEQ ID NO: 60); CDR2 containing the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL (amino acids 1-10 of SEQ ID NO: 61); And CDR3 containing the amino acid sequence GGSLSR (SEQ ID NO: 62). 11. The bispecific binding molecule of claim 10, wherein the human serum albumin ISVD contains the amino acid sequence EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSA (SEQ ID NO: 59). 12. A polynucleotide encoding a binding molecule according to any one of claims 1-11. 13. A vector containing a polynucleotide according to claim 12 for expressing a binding molecule in a host cell. 14. A host cell containing a polynucleotide according to claim 12 or a vector according to claim 13. 15. The method of obtaining a binding molecule according to claim 1, including (a) introducing a polynucleotide encoding a binding molecule according to claim 1 into the host cell; (b) culturing the host cell in a medium under conditions favorable for the expression of said binding molecule from said polynucleotide; and (c) purifying the binding molecule from said host cell and/or said medium. 16. Binding molecule obtained by the method according to claim 15. 17. A method for preventing the binding of LAG3 to MHC class II, comprising contacting said LAG3 with a binding molecule according to any one of claims 1 or 2 or a bispecific binding molecule according to any one of claims 4, 5 or 8-11. 18. A method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of a binding molecule according to any one of claims 1-9. - 104 -