Methods and Compositions

Abstract

The present invention relates to a method for selecting an immunotherapeutic agent for treating a disease in an individual and to the treatment of the disease by administering the immunotherapeutic agent or the cognate peptide antigen of the immunotherapeutic agent. The present invention also relates to an antigen-binding molecule that binds to the peptide antigen.

Description

[Technical field]

[0001] The present invention relates to a method for selecting an immunotherapeutic agent for treating a disease in an individual and to the treatment of the disease by administering the immunotherapeutic agent or the cognate peptide antigen of the immunotherapeutic agent. The present invention also relates to an antigen-binding molecule that binds to the peptide antigen. [Background technology]

[0002] Allogeneic stem cell / immune cell transplantation (allo-SCT) is the most established and commonly used cancer cell immunotherapy. Allo-SCT has been in routine clinical use since the 1960s. More than 20,000 allo-SCTs are performed annually worldwide, primarily for the highest-risk blood cancers, such as acute myeloid leukemia (AML).

[0003] In allo-SCT, blood stem cells and immune cells from a healthy individual are transplanted into the patient. Allo-SCT is a highly toxic treatment that is generally limited to patients under 65 years of age. Nevertheless, the procedure-related mortality rate of allo-SCT is 10-40%, and approximately 20-40% of patients die from disease recurrence. Only 30-70% of patients survive and are cured after allo-SCT.

[0004] Although many aspects of allo-SCT are not fully understood, it is known that the curative effect of allo-SCT depends on the alloreactivity of donor T cells against cancer cells. In the case of AML, this alloreactivity is known as graft-versus-leukemia (GvL). It is desirable to understand the underlying mechanisms of graft-versus-disease responses such as GvL so that we can maximize the chances of a favorable outcome and improve the safety of allo-SCT.

[0005] A key question in understanding the fundamental mechanisms of graft-versus-disease (e.g., GvL) responses is how donor T cells that mediate the curative response recognize tumor cells as targets. T cells use their T cell receptors (TCRs) to scan target cells for peptide antigens complexed with MHC molecules on their surface. However, the antigen-MHC complexes on recipient tumor cells that are recognized by donor T cells remain to be characterized. Uncovering such characteristics is crucial for understanding curative graft-versus-disease immune responses and for distinguishing between antigen-MHC complexes that contribute to such responses and those that induce pathogenic graft-versus-host disease (GvHD). Summary of the Invention

[0006] The present invention relates to defining the antigenic basis of beneficial graft-versus-disease and pathogenic graft-versus-host disease (GvHD) immune responses. Identifying antigens involved in the graft-versus-disease immune response allows for the selection of immunotherapeutic agent(s) specific for antigens presented on diseased cells. Identifying antigens involved in the GvHD immune response allows for the selection of immunotherapeutic agent(s) that do not target these antigens, thereby alleviating GvHD.

[0007] More specifically, the inventors have demonstrated that target cells in diseased individuals are capable of binding to MHC molecules and present peptide antigens on their surface that are encoded by germline diversity in diseased individuals compared to non-diseased individuals. The inventors have also identified antigen-binding molecules that bind to the peptide antigens and can be used to immunotherapy the disease. The inventors have also demonstrated that certain peptide antigens are present on the surface of normal, non-diseased cells, and that targeting these peptide antigens can induce pathogenic graft-versus-host disease responses.

[0008] Thus, the present invention provides a method for selecting an immunotherapeutic agent for treating a disease in an individual, comprising: (a) identifying a peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual, and optionally not present on the surface of a non-target cell in the individual; and (b) selecting an immunotherapeutic agent comprising an antigen-binding molecule that binds to the peptide antigen.

[0009] The present invention also provides - a method of treating a disease in an individual, comprising administering to the individual an immunotherapeutic agent comprising an antigen-binding molecule that binds to said peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual, and optionally not present on the surface of a non-target cell in the individual; - a method of treating a disease in an individual, comprising administering to the individual a composition comprising a peptide antigen, thereby inducing an immune response specific to the peptide antigen, wherein the peptide antigen (i) is encoded by germline diversity in the individual, (ii) is capable of binding to an MHC molecule, and (iii) is present on the surface of target cells in the individual, and optionally is not present on the surface of non-target cells in the individual; - an immunotherapeutic agent for use in a method of treating a disease in an individual, the method comprising administering the immunotherapeutic agent to the individual, the immunotherapeutic agent comprising an antigen-binding molecule that (i) is encoded by germline diversity in the individual, (ii) is capable of binding to an MHC molecule, and (iii) binds to a peptide antigen that is present on the surface of target cells in the individual, and optionally is not present on the surface of non-target cells in the individual; - a composition comprising a peptide antigen for use in a method of treating a disease in an individual, the method comprising administering to the individual the peptide antigen, thereby inducing an immune response specific to the peptide antigen, the peptide antigen (i) being encoded by germline diversity in the individual, (ii) being capable of binding to an MHC molecule, and (iii) being present on the surface of target cells in the individual, and optionally being absent on the surface of non-target cells in the individual; - an antigen-binding molecule that is (i) encoded by germline diversity in an individual, (ii) capable of binding to an MHC molecule, and (iii) that binds to a peptide antigen that is present on the surface of a target cell in the individual, and optionally is not present on the surface of a non-target cell in the individual; - a peptide antigen that is (i) encoded by germline variation in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual, and optionally not present on the surface of a non-target cell in the individual; - a polynucleotide encoding an antigen-binding molecule or peptide antigen of the invention; and - A vector comprising a polynucleotide of the invention to provide. [Brief description of the drawings]

[0010] [Figure 1] Exome diversity mismatched between patients and their stem cell transplant donors in 15 patient-donor pairs. a) Unrelated transplants harbor a higher number of mismatched germline variants compared to sibling transplants. b) 90.2% of variants were single nucleotide variations (SNVs), with missense being the most common finding in coding sequences (91.06%). [Diagram 2]Predicted HLA binding peptides in 15 patient-donor pairs. X-axis is individual patient. Y-axis is predicted number of HLA binding epitopes with germline variants. a) Predicted number of patient-donor mismatched HLA class I binding epitopes per patient. High affinity binders (rank affinity <0.5%) and low affinity binders (rank affinity 1.5-2%). b) Number of patient-donor mismatched HLA class II binding peptides per patient. High affinity binders (IC50 <150nM) and low affinity binders (IC50 150-500nM). [Diagram 3] Characterization of GvL T cell responses. a) IFNγ ELISpot results after stimulation of post-transplant PB with GvL peptide + / - HLA pan class I (W6 / 32), pan class II (Tue39), HLA-DR (L243), -DQ (Tue169), and -DP (B7 / 21) blocking antibodies. Negative control was no peptide (DMSO). b) Flow cytometry with intracellular cytokine staining showing % of T cells positive for IFNγ, TNFα, CD107a after peptide treatment vs. untreated. [Figure 4] AMADEUS trial overview and sample collection. a) Trial overview. b) Sample collection. Arrowheads indicate time points when samples were taken according to the trial protocol. (AML: acute myeloid leukemia; MDS: myelodysplasia; MRD: minimal residual disease; allo-SCT: allogeneic stem cell transplant). [Diagram 5]Expression of PADI4 in normal tissues and cancer. a) Graph showing RNAseq data for PADI4 in 37 normal human tissues (data taken from Human Protein Atlas (v18.1); Uhlen et al. Science 2015). TPM = transcripts per million reads. PADI4 SNPs have an allele frequency of 4.27%, are present in about 8% of the population, and are either homozygous or heterozygous. The graph shows that in normal tissues, PADI4 expression is highest in hematopoietic tissues (bone marrow and spleen). b) Graph showing RNAseq data for PADI4 in 21 human tumors (data taken from Human Protein Atlas (v18.1); Uhlen et al. Science 2017). c) Graph showing RNAseq data for PADI4 in 15 primary AML (acute myeloid leukemia) samples. [Figure 6] Identification of antigen-specific T cell receptors. The following protocol was used to sequence TCRs recognizing peptide 1 (SEQ ID NO:1) or peptide 2 (SEQ ID NO:2): 1. Culture post-transplant blood with peptide (identified from IFNγ ELISpot screening) to enrich for antigen-specific T cells. 2. FACS sort activated (IFNγ+) and control (IFNγ-) T cells after peptide stimulation. 3. Single-cell RNA sequencing with V(D)J enrichment. In peptide-activated (i.e. CD4+IFNγ+) T cells, two clones expand. The two expanded clones (CD4+IFNγ+) are putative antigen-specific T cells. The thick black bands (labeled 56.9% in the left panel and 11.0% in the right panel represent the sum of clonotypes with frequencies less than 0.1%). [Figure 7] Cytokine / activation markers in putative antigen-specific T cells. Single-cell RNA sequencing of FACS-sorted activated (IFNγ+) and control (IFNγ-) T cells shows that putative antigen-specific T cells express multiple cytokines (IFNγ, TNFα) and activation markers (FASLG, IL2RA, TNFRSF9, GZMB) in response to peptide stimulation. [Figure 8] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX289 peptide 52, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, two clones expanded (27.3% and 10.7%). These clones were present at lower frequencies in the control (IFNγ-) repertoire. [Figure 9] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX289 peptide 40, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, one clone expanded highly (85.2%). This clone was not present in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 52 cultures). [Figure 10] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX289 overlapping peptide 73 / 74, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, two clones were abundant and one was less abundant. These clones were not present in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 52 cultures). [Figure 11] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX289 peptide 221, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, two clones were abundant. These clones were not present in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 52 cultures). [Figure 12]Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX289 overlapping peptide 75 / 205, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, three clones expanded. These clones were not present in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 52 cultures). [Figure 13] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX802 overlapping peptide 321 / 322, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, two clones were abundant: clone 1 was present in the control (IFNγ-) repertoire, whereas clone 2 was absent. [Figure 14] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX628 peptide 80 / 81, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, two clones expanded (55.1% and 13.5%). These clones were present at lower frequencies in the control (IFNγ-) repertoire. [Figure 15] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX747 overlapping peptide 157 / 158, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, multiple clones of moderate size expanded. All were present at lower frequencies in the control (IFNγ-) repertoire. [Figure 16] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX885 overlapping peptide 559 / 560, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, one clone expanded highly (96.5%). This clone was also frequently present in the control (IFNγ-) repertoire. (Note - the negative control population was sorted from the same culture as activation) [Figure 17]Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX885 overlapping peptide OX628-308 / 309, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, one clone was abundant (76.1%). This clone was present at low frequency in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 559 / 560 cultures). [Figure 18] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing the OX993 overlapping peptide 413 / 414, the protocol described in Example 5 was used. Four clones expanded after peptide stimulation and IFNγ capture compared to the control (IFNγ-) repertoire. [Figure 19] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX1149 peptide 190, the protocol described in Example 5 was used. Three clones expanded after peptide stimulation and IFNγ capture compared to the control (IFNγ-) repertoire. [Figure 20] Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX1149 peptide 290, the protocol described in Example 5 was used. After peptide stimulation and IFNγ capture, three clones expanded. These clones were not present in the control (IFNγ-) repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 190 cultures). [Figure 21] Identification of antigen-specific T cell receptors. To determine the sequence of the TCR that recognizes OX289 peptide 284, the protocol described in Example 5 was used. Dominant clones in the negative control (IFNγ-) repertoire were more prevalent in the activated repertoire (96.0% vs. 65.1%). [Figure 22]Identification of antigen-specific T cell receptors. To sequence the TCR recognizing OX289 peptide 9, the protocol described in Example 5 was used. Dominant clones in the negative control (IFNγ-) repertoire were more prevalent in the activated repertoire (91.1% vs. 65.1%). (Note - the negative control was sorted as the IFNγ- population from peptide 284 cultures, not peptide 9 cultures). [Diagram 23] Identification of antigen-specific T cell receptors. To sequence the TCR that recognizes OX289 peptide 67, the protocol described in Example 5 was used. The two most abundant clones in the activation repertoire were absent from the control repertoire. (Note - the negative control was sorted as the IFNγ- population from peptide 284 cultures). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present inventors have identified for the first time peptide antigens targeted by immune responses in graft-versus-disease (such as graft-versus-cancer or graft-versus-leukemia, GvL) and graft-versus-host disease. The present inventors have also identified cognate antigen-binding molecules, such as T cell receptors (TCRs), that recognize peptide antigens targeted by the graft-versus-disease response and can be used to provide immunotherapy for the disease.

[0012] More specifically, the inventors have demonstrated that graft-versus-disease and graft-versus-host disease immune responses are primarily specific for peptide antigens that are encoded by the germline diversity of the diseased individual and are present on the surface of cells targeted by allogeneic hematopoietic stem cell transplantation.

[0013] With knowledge of the antigenic basis of the graft-versus-disease immune response, it is possible to select donor-patient pairs for allo-SCT that maximize the chances of a successful therapeutic outcome. It is also possible to provide immunotherapeutics that contain antigen-binding molecules (such as T cell receptors, T cell receptor fragments, chimeric antigen receptors (CARs), antibodies, antibody fragments, or bispecific T cell engagers (BiTEs)) that mimic the antigen specificity of donor T cells that can mount an effective graft-versus-disease immune response. In this way, the graft-versus-disease immune response normally associated with allo-SCT can be harnessed for use in off-the-shelf or autologous therapeutic approaches. It is also possible to prime immune cells present in the patient to mount a response against disease-associated target cells by administering the relevant peptide antigen as a vaccine. Any of these approaches may be used in combination.

[0014] Knowledge of the antigenic basis of the graft-versus-host immune response can also be used to select donor-patient pairs for allo-SCT. In particular, donor cells that recognize peptide antigens present on non-target cells (such as normal cells in healthy tissues) can be avoided. Immunotherapeutic agents that contain antigen-binding molecules that mimic the antigenic specificity of such donor cells can be avoided as well, as can priming immune cells present in the patient and specific for graft-versus-host peptide antigens.

[0015] To clarify the antigenic basis of graft-versus-host disease and graft-versus-host disease immune responses, the inventors compared the sequences of genomic DNA of patients and donors to identify germline-encoded variants that are mismatched between patients and donors. In an exemplary embodiment, the patient is an AML patient and the donor is an AML-free donor. However, the types of antigens identified as the basis of the graft-versus-AML response (i.e., MHC-restricted peptide antigens encoded by germline diversity in the patient and present on the surface of target cells) may also be expressed by target cells associated with other diseases, such as other types of cancer. In addition, because the graft-versus-AML response targets germline diversity (e.g., not disease-specific neoepitopes or tumor-associated antigens), the peptide antigens may also be expressed by different types of target cells in different individuals, such as target cells derived from different tissues. For example, the same peptide antigen may be expressed on the surface of AML cells in one individual and on the surface of solid tumor cells in another individual.

[0016] Thus, the approach we have adopted has broad implications for the treatment of diseases such as cancer. This approach allows the selection of immunotherapeutic agents or peptide antigen vaccines that are effective against different individuals with different types of diseases, such as different types of cancer. Furthermore, a single immunotherapeutic agent with a single antigen specificity may be useful in treating different diseases in different individuals, each with a particular germline diversity, depending on the type of target cell on whose surface the peptide antigen is expressed. This may be particularly true for peptides encoded in germline diversity expressed by target cells from more than one patient, because they may be so-called "public" antigens that are present at high frequency in the population (e.g., frequency 0.05 or higher).

[0017] Various aspects of the invention are described in detail below.

[0018] How to Select an Immunotherapeutic Agent The present invention provides a method for selecting an immunotherapeutic agent for treating a disease in an individual, comprising: (a) identifying a peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual, and optionally not present on the surface of a non-target cell in the individual; and (b) selecting an immunotherapeutic agent comprising an antigen-binding molecule that binds to the peptide antigen. Preferably, the antigen-binding molecule binds to the peptide antigen when the peptide antigen is bound to an MHC molecule. That is, the antigen-binding molecule can bind to a peptide-MHC complex that contains the peptide antigen.

[0019] By identifying a peptide antigen and selecting an immunotherapeutic agent containing an antigen-binding molecule that binds to the peptide antigen, the possibility of successful treatment of a disease by administering the immunotherapeutic agent to an individual is maximized. In short, by matching the antigen-binding ability of the antigen-binding molecule contained in the immunotherapeutic agent to the antigen expression profile of the target cell, the probability that the antigen-binding molecule will bind to the target cell can be increased, thereby achieving treatment of the disease.

[0020] The absence of peptide antigens on the surface of non-target cells minimizes the risk of graft-versus-host disease resulting from administration of an immunotherapeutic agent to an individual, where the immunotherapeutic agent selectively binds to target cells but not to non-target cells, as described in more detail below.

[0021] Medicines and Treatment Methods The present invention provides a method of treating a disease in an individual, comprising administering to the individual an immunotherapeutic agent comprising an antigen-binding molecule that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) that binds to a peptide antigen present on the surface of target cells in the individual and, optionally, not present on the surface of non-target cells in the individual. The present invention also provides an immunotherapeutic agent for use in a method of treating a disease in an individual, comprising administering to the individual the immunotherapeutic agent, the immunotherapeutic agent comprising an antigen-binding molecule that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) that binds to a peptide antigen present on the surface of target cells in the individual and, optionally, not present on the surface of non-target cells in the individual.

[0022] The peptide antigen is present on the surface of the target cell because it is bound to an MHC molecule on the cell surface. After administration of the immunotherapeutic agent, the antigen-binding molecule specifically binds to the target cell via the peptide antigen. The binding can achieve treatment of the disease in various ways depending on the nature of the antigen-binding molecule and / or the immunotherapeutic agent. For example, the immunotherapeutic agent may include a T cell expressing a T cell receptor (TCR) as an antigen-binding molecule. Binding of the T cell receptor to the peptide antigen-MHC complex can trigger activation of the T cell, which can result in, for example, cytolysis of the target cell and / or production of cytokines harmful to the target cell, such as interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα). The immunotherapeutic agent may include an antigen-binding molecule including, for example, an antibody or an antibody fragment. When the antibody or antibody fragment binds to the peptide antigen, the target cell can be decorated with opsonins and marked for phagocytosis or destruction via the classical complement pathway. The antibody / antibody fragment can bind to a chemotherapeutic agent, so that the chemotherapeutic agent can be delivered to the target cell by binding the antibody or antibody fragment to the peptide antigen. Immunotherapeutic agents may include antigen-binding molecules, including, for example, bispecific T cell engagers (BiTEs). BiTEs can simultaneously bind (i) a peptide antigen and (ii) a molecule expressed on the surface of a T cell (e.g., CD3) to form a link between the target cell and the T cell that is independent of the antigen specificity of the T cell. Binding of the surface molecule on the T cell can activate the T cell, leading to, for example, cytolytic activity against the target cell.

[0023] The absence of peptide antigens on the surface of non-target cells minimizes the risk of graft-versus-host disease resulting from administration of an immunotherapeutic agent to an individual, where the immunotherapeutic agent selectively binds to target cells but not to non-target cells, as described in more detail below.

[0024] The step of administering an immunotherapeutic agent to an individual may, for example, include administering two or more immunotherapeutic agents to the individual, each of which comprises an antigen-binding molecule that binds to a peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual and optionally not present on the surface of a non-target cell in the individual. For example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more immunotherapeutic agents may be administered to the individual, each of which comprises an antigen-binding molecule that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual and optionally not present on the surface of a non-target cell in the individual. Each immunotherapeutic agent administered to the individual may, for example, comprise an antigen-binding molecule that binds to a different peptide antigen. Each different peptide antigen may be encoded by a different germline diversity in the individual.

[0025] The step of administering an immunotherapeutic agent to an individual may, for example, include administering a single immunotherapeutic agent to the individual, the single immunotherapeutic agent comprising two or more antigen-binding molecules that each bind to a peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual and optionally not present on the surface of a non-target cell in the individual. For example, a single immunotherapeutic agent may be administered to an individual, comprising three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more antigen-binding molecules that each bind to a peptide antigen that is (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of a target cell in the individual and optionally not present on the surface of a non-target cell in the individual. Each antigen-binding molecule included in the single immunotherapeutic agent may, for example, bind to a different peptide antigen. Each different peptide antigen may be encoded by a different germline diversity in the individual.

[0026] In another aspect, the invention provides a method of treating a disease in an individual, comprising administering to the individual a composition comprising a peptide antigen, thereby inducing an immune response specific to the peptide antigen, wherein the peptide antigen (i) is encoded by germline diversity in the individual, (ii) is capable of binding to an MHC molecule, and (iii) is present on the surface of target cells in the individual, and optionally is not present on the surface of non-target cells in the individual. The invention also provides a composition comprising a peptide antigen for use in a method of treating a disease in an individual, comprising administering to the individual, thereby inducing an immune response specific to the peptide antigen, wherein the peptide antigen (i) is encoded by germline diversity in the individual, (ii) is capable of binding to an MHC molecule, and (iii) is present on the surface of target cells in the individual, and optionally is not present on the surface of non-target cells in the individual.

[0027] After administration of the peptide antigen, components of the individual's own immune system may be primed to mount an immune response against the target cells. For example, administration of the peptide antigen may stimulate proliferation of T cells and / or B cells specific to the peptide antigen. Administration of the peptide antigen may also stimulate activation of T cells and / or B cells specific to the peptide antigen. In this way, T cell and / or B cell responses against the target cells may be induced. The absence of the peptide antigen on the surface of non-target cells minimizes the risk of graft-versus-host disease due to a primed immune response.

[0028] The step of administering a peptide antigen to an individual may, for example, comprise administering two or more peptide antigens to the individual, each peptide antigen being (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. For example, the individual may be administered three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more peptide antigens, each of which being (i) encoded by germline diversity in the individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. Each peptide antigen administered to the individual may, for example, be encoded by a different germline diversity in the individual. The peptide antigens may be included in the same composition.

[0029] The method of treating a disease in an individual may further comprise administering to the individual immune cells specific for the peptide antigen. In other words, (i) the peptide antigen and (ii) the peptide antigen-specific immune cells may be administered to the individual. After administration of the peptide antigen, the peptide antigen-specific cells may be primed to mount an immune response against the target cells. This may facilitate therapeutic benefit when components of the individual's own immune system are tolerant to the peptide antigen. The peptide antigen and the peptide antigen-specific immune cells may be included in the same composition. The absence of the peptide antigen on the surface of non-target cells minimizes the risk of graft-versus-host disease due to a primed immune response.

[0030] As described above, when administering multiple peptide antigens to an individual, the method may include administering immune cells specific for each peptide antigen to the individual. The administered immune cells may, for example, include separate immune cells each specific for one of the peptide antigens. The administered immune cells may, for example, include one or more immune cells each specific for multiple peptide antigens. For example, when administering peptide antigen X and peptide antigen Y to an individual, the method may include administering (a) immune cells specific for peptide antigen X and immune cells specific for peptide antigen Y, or (b) immune cells specific for both peptide antigen X and peptide antigen Y, individually. In either case, the peptide antigens and the immune cells specific for the peptide antigens may be included in the same composition.

[0031] Alternatively, the peptide antigen and the peptide antigen-specific immune cells may be included in different compositions. In this case, the composition comprising the peptide antigen and the composition comprising the peptide antigen-specific immune cells may be administered together to the individual. That is, the composition comprising the peptide antigen and the composition comprising the peptide antigen-specific immune cells may be administered to the individual substantially simultaneously. The composition comprising the peptide antigen and the composition comprising the peptide antigen-specific immune cells may be administered separately to the individual. That is, the composition comprising the peptide antigen and the composition comprising the peptide antigen-specific immune cells may be administered to the individual at different times. For example, the method may include administering to the individual (i) a composition comprising the peptide antigen followed by (ii) a composition comprising the peptide antigen-specific immune cells. Preferably, the method may include administering to the individual (i) a composition comprising the peptide antigen-specific immune cells followed by (ii) a composition comprising the peptide antigen.

[0032] The peptide antigen-specific immune cells may include, for example, T cells, B cells, NK cells, NKT cells, macrophages, and / or monocytes. Preferably, the peptide antigen-specific cells include T cells and / or B cells. More preferably, the peptide antigen-specific immune cells include T cells. The peptide antigen-specific immune cells may be naturally occurring. For example, the peptide antigen-specific immune cells may be obtained from a donor. The donor may be, for example, a donor with a genome that does not include germline diversity. Preferably, the donor is disease-free. The peptide antigen-specific immune cells may be, for example, genetically engineered cells. The genetically engineered immune cells may be autologous or allogeneic to the individual. The immune cells may be genetically engineered to include a receptor that confers specificity for the peptide antigen. For example, the immune cells may include a T cell receptor or a chimeric antigen receptor (CAR) specific for the peptide antigen.

[0033] The immune response induced by administration of the peptide antigen may be an adaptive immune response. For example, the immune response may include a T cell response, a B cell response, and / or a NKT cell response. Preferably, the immune response includes a T cell response. The T cell response may include a CD4+ cell response. The T cell response may include a CD8+ cell response. Preferably, the MHC molecule capable of binding the peptide antigen is an MHC class II molecule, and the T cell response includes a CD4+ T cell response. Preferably, the MHC molecule capable of binding the peptide antigen is an MHC class I molecule, and the T cell response includes a CD8+ T cell response.

[0034] CD4+T cells and / or CD8+T cells involved in T cell responses may have cytolytic effector function. CD4+T cells and / or CD8+T cells may express markers of cytolytic effector function. For example, CD4+T cells and / or CD8+T cells may express one or more of perforin, granzyme A, granzyme B, Fas, FasL, and TRAIL. CD4+T cells and / or CD8+T cells involved in T cell responses may express CD107a, a degranulation marker.

[0035] CD4+ T cells and / or CD8+ T cells involved in a T cell response may secrete one or more cytokines. For example, CD4+ T cells and / or CD8+ T cells may secrete interferon gamma (IFNγ). CD4+ T cells and / or CD8+ T cells may secrete tumor necrosis factor alpha (TNFα).

[0036] The immune response induced by administration of a peptide antigen can be an innate immune response. For example, the immune response can include an NK cell response, a macrophage response, a dendritic cell response, or a monocyte response.

[0037] The method may further include administering an adjuvant to the individual. The adjuvant may include, for example, one or more of incomplete Freund's adjuvant (IFA), Montanide, aluminum adjuvant (Alum), microparticles, nanoparticles, Toll-like receptor (TLR) agonists, cytokines, or antibodies. The aluminum adjuvant may include aluminum hydroxide and / or aluminum phosphate. The microparticles or nanoparticles may include, for example, liposomes, synthetic polymers (such as polystyrene, poly(lactide-co-glycolide) PLG, poly(lactic acid) PLA, and / or PLGA), natural polymers (such as gelatin, collagen, and / or chitosan), and / or carbon nanotubes. The TLR agonists may include, for example, Pam3CSK4, Poly-ICLC, MPLA, Imiquimod, and / or CpG. The cytokines may include, for example, IL-2, GM-CSF, IFN, or CDN. The antibody may, for example, comprise an anti-PD1 antibody or an anti-CTLA-4 antibody. Preferably, the adjuvant is administered together with the peptide antigen. That is, the adjuvant and the peptide antigen may be administered to an individual substantially simultaneously. For example, the adjuvant and the peptide antigen may be included in the same composition. In other words, the composition comprising the peptide antigen may further comprise an adjuvant.

[0038] disease The disease may be any disease, such as a disease that causes or results from the presence of unwanted cells in an individual. The unwanted cells may, for example, be involved in the pathogenesis of the disease. The unwanted cells may, for example, be harmful to the individual. The immunotherapeutic agent may target the unwanted cells to impair their function or kill them, thereby treating the disease. The unwanted cells may express MHC class II. The unwanted cells may express MHC class I. The unwanted cells may be target cells.

[0039] The disease may be, for example, cancer. In this case, the undesirable cells may be cancer cells. The cancer may be, for example, anal cancer, bile duct cancer (cystic duct cancer), bladder cancer, blood cancer, bone cancer, intestinal cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endocrine tumors, Ewing's sarcoma, eye cancer (e.g., ocular melanoma), fallopian tube cancer, gallbladder cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumors, ovarian cancer, esophageal cancer, pancreatic cancer, penile cancer, primary peritoneal cancer, prostate cancer, pseudomyxoma peritonei, skin cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, tracheal cancer, cancer of unknown primary, vaginal cancer, vulvar cancer, or endometrial cancer. The leukemia is preferably acute lymphoblastic leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia, or chronic myelogenous leukemia. The lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma. The cancer may be, for example, acute myeloid leukemia (AML). The cancer cell may be, for example, an AML cell.

[0040] The disease may be, for example, an autoimmune disease. In this case, the unwanted cells may be immune cells. The immune cells may be, for example, lymphocytes, such as T cells or B cells. The lymphocytes may contain antigen receptors specific for self-antigens. The autoimmune disease may be, for example, alopecia areata, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), autoimmune juvenile idiopathic arthritis, celiac disease, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, uveitis, or vitiligo.

[0041] The disease may be, for example, an allergic disease. In this case, the undesirable cell may be an immune cell. The immune cell may be, for example, a lymphocyte, such as a T cell or a B cell. The lymphocyte may include an antigen receptor specific for an allergen. The allergic disease may be, for example, atopic dermatitis, allergic airway inflammation, or perennial allergic rhinitis.

[0042] The disease may be, for example, a fibrotic disease. A fibrotic disease is a disease in which inflammation and / or tissue damage causes fibrosis. In this case, the unwanted cells may be fibroblasts. The unwanted cells may be, for example, lymphocytes, such as T cells or B cells. The fibrotic disease may be, for example, pulmonary fibrosis (such as cystic fibrosis or idiopathic pulmonary fibrosis), myocardial fibrosis (such as interstitial fibrosis or replacement fibrosis), liver cirrhosis, bridging fibrosis of the liver, glial scar, arteriosclerosis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, retroperitoneal fibrosis, scleroderma, systemic sclerosis, or adhesive capsulitis.

[0043] individual Preferably, the individual is a human individual. Alternatively, the individual may be a non-human mammal. For example, the individual may be a pet mammal (such as a cat, dog, horse, rabbit, or guinea pig), a commercially farmed mammal (such as a cow, sheep, goat, or pig), or a laboratory mammal (such as a mouse or rat). The individual may be a young, juvenile, or adult. The individual may be known to have a disease or may be suspected to have a disease. The terms "individual" and "patient" may be used interchangeably.

[0044] Peptide antigens The above methods, medicaments, and medical uses include peptide antigens that are (i) encoded by germline diversity in an individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. The present invention further provides such peptide antigens. That is, the present invention further provides peptide antigens (such as isolated peptide antigens) that are (i) encoded by germline diversity in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. Any of the aspects described above in relation to the methods, medicaments, and medical uses of the present invention may also be applied to the peptide antigens of the present invention.

[0045] The term "peptide" relates to a short chain of amino acids linked by peptide (-CO-NH-) bonds. A peptide antigen may comprise or consist of, for example, 10 to 20 amino acids, for example, 11 to 19 amino acids, 12 to 18 amino acids, 13 to 17 amino acids, or 14 to 16 amino acids. Preferably, a peptide antigen comprises or consists of 13 to 17 amino acids. A peptide antigen may comprise, for example, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids. A peptide antigen may consist of, for example, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0046] A peptide antigen may comprise or consist of an epitope. An epitope may be, for example, a CD4+ T cell epitope, a B cell epitope, or an antibody epitope. A CD4+ T cell epitope is a peptide that (i) can be presented by an MHC molecule and (ii) can bind to a T cell receptor (TCR) present on a CD4+ T cell. Preferably, upon TCR binding, the CD4+ T cell is activated. Activation of CD4+ T cells may lead to increased proliferation, cytokine production, and / or cytotoxicity. A B cell epitope is a peptide that can bind to a B cell receptor (BCR) present on a B cell. Preferably, upon BCR binding, the B cell is activated and / or matures. Activation of B cells may lead to increased proliferation and / or antibody production. An antibody epitope is a peptide that can bind to a paratope of an antibody.

[0047] A variety of peptide antigens have been identified in the Examples, including: LTISLLDTFNLELPEAVVFQ (SEQ ID NO: 1) ISLLDTFNLELPEAVVFQDS (SEQ ID NO: 2) SLLDTFNLELPEAVVF (SEQ ID NO: 3) LDTFNLELPEAVVFQ (SEQ ID NO: 4) AASCEPLASVLRAKLTSRSS (SEQ ID NO: 380) GAGPDPLRLHGHLPVRTSCP (SEQ ID NO: 381) TQRSVLLCKVVGARGVGKSA (SEQ ID NO: 382) KVVGARGVGKSAFLQAFLGR (SEQ ID NO: 383) GQKSPRFRRVSCFLRLGRST (SEQ ID NO: 384) RFRRVSCFLRLGRSTLLELE (SEQ ID NO: 385) ALAFLLLISIAANLSLLLSR (SEQ ID NO:386) EVQDCLKQLMMSLLQLYRFS (sequence number 387) GYSPSLHILAIGTRSGAIKL (SEQ ID NO: 388) VATFPVYTMVAIPIVCKD (SEQ ID NO: 389) PPLYRQRYQFIKNLVDQHEP (SEQ ID NO: 390) PLYRQRYQFIKNLVDQHEPK (SEQ ID NO: 391) VSRPELLRESISAFLVPMPT (SEQ ID NO: 392) TDRALQNKSISAFLVPMPTP (SEQ ID NO: 393) NPLSPYLNVDPRYLVQDT (SEQ ID NO: 394) EPPVDICLSKAISSSLKGFL (SEQ ID NO: 395) GETGMFSLSTIRGHQYATY (SEQ ID NO: 396) MNYVSKRLPFAARLNTPMGP (SEQ ID NO: 397) LGSLGLIFALTLNRHKYPLN (SEQ ID NO: 398) LGLIFALTLNRHKYPLNLYL (SEQ ID NO: 399) APISLSSFFNVSTLEREVTD (sequence number 400) LELGAGTGLASIIAATMART (SEQ ID NO: 401) AGTGLASIIAATMARTVYCT (SEQ ID NO: 402) VPREYVRALNATKLERVFAK (SEQ ID NO: 403) LHRDKALLKRLLKGMQKKRP (SEQ ID NO: 404) KALLKRLLKGMQKKRPSDVQ (SEQ ID NO: 405) ITVQTVYVQHLITFLDRPIQ (sequence number 406) QTVYVQHLITFLDRPIQMCC (sequence number 407) PGLISMFSSSQELGAALAQL (sequence number 408) WRVRIALALKGIDYETVPIN (SEQ ID NO: 409) VRIALALKGIDYETVPINLI (SEQ ID NO: 410) DRAEKFNRGIRKLGITPEGQ (SEQ ID NO: 411) EKFNRGIRKLGITPEGQSYL (sequence number 412).

[0048] The peptide antigen may comprise or consist of SEQ ID NO: 3. The peptide antigen may comprise, for example, 10 to 20 (e.g., 11 to 19, 12 to 18, 13 to 17, or 14 to 16) amino acids and may comprise SEQ ID NO: 3. For example, the peptide antigen may comprise or consist of SEQ ID NO: 1. The peptide antigen may comprise or consist of SEQ ID NO: 4. The peptide antigen may comprise, for example, 10 to 20 (e.g., 11 to 19, 12 to 18, 13 to 17, or 14 to 16) amino acids and may comprise SEQ ID NO: 4. For example, the peptide antigen may comprise or consist of SEQ ID NO: 2. The peptide antigen may comprise any one of SEQ ID NOs: 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, and 412. The peptide antigen may consist of any one of SEQ ID NOs: 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, and 412.

[0049] target cell The peptide antigen is present on the surface of a target cell in an individual. The target cell may express MHC class II. The target cell may express MHC class I. The target cell may be an unwanted cell. The target cell may be involved in the pathogenesis of a disease. The target cell may be harmful to the individual.

[0050] The target cell may be, for example, a cancer cell. Cancer may be, for example, anal cancer cells, bile duct cancer (cholangiocarcinoma) cells, bladder cancer cells, blood cancer cells, bone cancer cells, intestinal cancer cells, brain cancer cells, breast cancer cells, colorectal cancer cells, cervical cancer cells, endocrine tumor cells, eye cancer (such as ocular melanoma) cells, fallopian tube cancer cells, gallbladder cancer cells, head and / or neck cancer cells, Kaposi's sarcoma cells, kidney cancer cells, laryngeal cancer cells, leukemia cells, liver cancer cells, lung cancer cells, lymph node cancer cells, lymphoma ... The cancer cells may be ovarian cancer cells, melanoma cells, mesothelioma cells, myeloma cells, neuroendocrine tumor cells, ovarian cancer cells, esophageal cancer cells, pancreatic cancer cells, penile cancer cells, primary peritoneal cancer cells, prostate cancer cells, pseudomyxoma peritonei cells, skin cancer cells, small intestine cancer cells, soft tissue sarcoma cells, spinal tumor cells, gastric cancer cells, testicular cancer cells, thymic cancer cells, thyroid cancer cells, tracheal cancer cells, cancer of unknown primary, vaginal cancer cells, vulvar cancer cells, or endometrial cancer cells. The leukemia may be acute lymphoblastic leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia, or chronic myeloid leukemia. The lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma. Preferably, the cancer cells are AML cells.

[0051] The target cell may be, for example, an immune cell. For example, the immune cell may be a lymphocyte, such as a T cell or a B cell. The lymphocyte may contain an antigen receptor specific for an autoantigen. The specificity for an autoantigen may cause, for example, alopecia areata, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), autoimmune juvenile idiopathic arthritis, celiac disease, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, uveitis, or vitiligo. Lymphocytes may contain antigen receptors specific for allergens, which may cause, for example, atopic dermatitis, allergic airway inflammation, or perennial allergic rhinitis.

[0052] The target cell may be, for example, a fibroblast.

[0053] The peptide antigen is present on the surface of the target cell. If any method for determining surface presence shows that the target cell presents detectable levels of the peptide antigen on its surface, the peptide antigen may be considered to be present on the surface of the target cell. Methods for determining surface presence are known in the art. Surface expression can be determined, for example, by immunoprecipitating MHC molecules from the target cell and mass spectrometry of the eluted MHC-bound peptides. Surface presence can be determined, for example, by flow cytometry. For example, flow cytometry can be used to measure the mean fluorescence intensity (MFI) of the peptide antigen. MFI measures intensity, and is measured in watts per square meter of time-averaged energy flux.

[0054] non-target cells As noted above, the peptide antigen is present on the surface of target cells in an individual with a disease. In any of the aspects described herein, the peptide antigen is preferably not present on the surface of non-target cells in the individual.

[0055] Target cells are described in detail above. Non-target cells may be any endogenous cells in an individual that are not target cells. In other words, non-target cells may be any endogenous cells that (a) are not undesirable cells, (b) are not involved in the pathogenesis of a disease, and / or (c) do not harm the individual. Thus, non-target cells may be desirable cells. Non-target cells may be non-pathogenic cells. Non-target cells may be harmless cells. In other words, non-target cells may be healthy or normal cells. Non-target cells may be non-cancer cells, such as non-AML cells.

[0056] The non-target cells may express MHC class II. The non-target cells may express MHC class I.

[0057] The term "not present on the surface of non-target cells" may be used interchangeably with the term "not detectable on the surface of non-target cells". Thus, if any method for determining surface presence indicates that the peptide antigen is not detectable on the surface of non-target cells, the peptide antigen may be considered not present on the surface of non-target cells. Methods for determining surface presence (and thus, surface absence) are known in the art. Surface expression can be determined, for example, by immunoprecipitating MHC molecules from target cells and mass spectrometry of the eluted MHC-bound peptides. Surface presence can be determined, for example, by flow cytometry. For example, flow cytometry can be used to measure the mean fluorescence intensity (MFI) of the peptide antigen. MFI measures intensity, and is measured in watts per square meter of time-averaged energy flux.

[0058] The peptide antigen may not be present on the surface of a particular type of non-target cell. For example, the peptide antigen may not be present on the surface of non-target cells in a particular tissue. The peptide antigen may not be present on the surface of non-target cells in a particular organ. For example, the peptide antigen may not be present on the surface of non-target cells in the anus, bile duct, bladder, blood, bone, intestine, brain, breast, colon, rectum, cervix, endocrine organs, eye, fallopian tube, gallbladder, head, neck, kidney, larynx, liver, lung, lymph node, muscle, peripheral nerve, ovary, esophagus, pancreas, penis, peritoneum, prostate, skin, spinal cord, stomach, testes, thymus, thyroid, trachea, vagina, vulva, and / or endometrium, alone or in any combination.

[0059] The peptide antigen may not be present on the surface of any number or percentage of non-target cells in an individual. For example, the peptide antigen may not be present on the surface of all non-target cells. The peptide antigen may not be present on the surface of 70% or more of non-target cells in an individual. For example, the peptide antigen may not be present on the surface of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of non-target cells in an individual. In other words, the peptide antigen may be present on 1% or less, 2% or less, 3% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, or 30% or less of non-target cells in an individual. The peptide antigen may not be present on the surface of any percentage or number of non-target cells in a particular tissue. The peptide antigen may not be present on the surface of any percentage or number of non-target cells in a particular organ. For example, the peptide antigens, alone or in any combination, may not be present on the surface of any proportion or number of non-target cells in the anus, bile duct, bladder, blood, bone, intestine, brain, breast, colon, rectum, cervix, endocrine organs, eye, fallopian tube, gallbladder, head, neck, kidney, larynx, liver, lung, lymph node, muscle, peripheral nerve, ovary, esophagus, pancreas, penis, peritoneum, prostate, skin, spinal cord, stomach, testis, thymus, thyroid, trachea, vagina, vulva, and / or endometrium.

[0060] Germline diversity Peptide antigens are encoded by germline diversity in an individual. Germline diversity is the variation in genetic structure inherited from parents and can be passed on to offspring. Thus, peptide antigens are not encoded by somatic diversity specific to the target cell. In other words, peptide antigens are not neoantigens and do not contain neoepitopes. Peptide antigens are not tumor-specific antigens (TSAs).

[0061] Thus, the peptide antigen may be present on the surface of different types of target cells in different individuals, such as target cells derived from different tissues. For example, the peptide antigen may be present on the surface of AML cells in one individual and on the surface of solid tumor cells in another individual. Thus, a single immunotherapeutic agent may be used to treat different diseases in different individuals, each with germline diversity, depending on the type of target cell on whose surface the peptide antigen is expressed.

[0062] Preferably, the peptide antigen binds to an antigen-binding domain expressed by immune cells derived from a donor with a genome that does not contain germline diversity. Preferably, the donor is disease-free. The donor may be an actual or intended donor. The antigen-binding domain may be, for example, a T cell receptor, a B cell receptor, an antibody, or an antibody fragment. The antibody fragment may be a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a single chain variable fragment (scFv), a scFv-Fc. The immune cell may be, for example, a T cell, a B cell, or a plasma cell. Preferably, the immune cell is a T cell. The immune cell may recognize the peptide antigen as "foreign" or "non-self". The peptide antigen may be immunogenic to the donor. The peptide antigen may be a non-major histocompatibility antigen.

[0063] In other words, germline diversity may be diversity to the genome of a donor or a prospective donor. Germline diversity may result in polymorphisms between an individual and a donor. Polymorphisms may exist, for example, between a protein or peptide in an individual and a corresponding protein or peptide in a donor. Germline diversity may cause a peptide antigen to be immunogenic to the donor. Germline diversity may code for a non-major histocompatibility antigen.

[0064] Germline diversity may include substitutions, insertions, and / or deletions of one or more nucleotides relative to the donor genome. For example, germline diversity may include substitutions of two or more, three or more, four or more, or five or more nucleotides relative to the donor genome. Germline diversity may include insertions of two or more, three or more, four or more, or five or more nucleotides relative to the donor genome. Germline diversity may include deletions of two or more, three or more, four or more, or five or more nucleotides relative to the donor genome. The amino acid substitutions may be, for example, conservative amino acid substitutions. The amino acid substitutions may be, for example, non-conservative amino acid substitutions.

[0065] Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid being replaced. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 main amino acids defined in Table 1 below. When amino acids have similar polarity, this can also be determined by referring to the hydropathy scale of amino acid side chains in Table 2.

[0066] [Table 1]

[0067] [Table 2]

[0068] MHC binding The peptide antigen is capable of binding to an MHC molecule.

[0069] The MHC molecule may be an MHC class I molecule. The MHC class I molecule may be a human MHC class I molecule, i.e., the MHC class I molecule may be an HLA class I molecule.

[0070] The peptide antigen may be capable of binding to one or more HLA class I molecules. For example, the peptide antigen may be capable of binding to one or more of (i) HLA-A molecules, (ii) HLA-B molecules, and (iii) HLA-C molecules. For example, the peptide antigen may be capable of binding to (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii).

[0071] The MHC molecule may preferably be an MHC class II molecule. The expression of MHC class II molecules has traditionally been limited to professional antigen-presenting cells. However, upregulation of MHC class II expression has been demonstrated in some target cells associated with certain diseases, such as AML, in patients. The MHC class II molecule may be a human MHC class II molecule. That is, the MHC class II molecule may be an HLA class II molecule.

[0072] The peptide antigen may be capable of binding to one or more HLA class II molecules. For example, the peptide antigen may be capable of binding to one or more of: (i) HLA-DP molecules, (ii) HLA-DM molecules, (iii) HLA-DOA molecules, (iv) HLA-DOB molecules, (v) HLA-DQ molecules, and (vi) HLA-DR molecules. For example, the peptide antigen may be capable of binding to one or more of: (i);(ii);(iii);(iv);(v);(vi);(i) and (ii);(i) and (iii);(i) and (iv);(i) and (v);(i) and (vi);(ii) and (iii);(ii) and (iv);(ii) and (v);(ii) and (vi);(iii) and (iv);(iii) and (v);(iii) and (vi);(iv) and (v);(iv) and (vi);(v) and (vi);(i), (ii), and (iii);(i), (ii), and (iv);(i), (ii), and (v);(i), (ii), and (vi);(i), (iii), and (iv);(i), (iii), and (v);(i), (iii), and (vi);(i), (iv), and (v);(i), (iv), and (vi);(i), (v), and (vi);(ii), (iii), and (iv);(ii), (iii), and (v);(ii), (iii), and (vi);(ii), (iv), and (v);(ii), (iv), and (vi);(ii), (v), and (vi);(iii), (iv), and (v);(iii), (iv), and (vi);(iii), (v), (vi);(iv), (v), and (vi);(i), (ii), (iii), and (iv);(i), (ii), (iii), and (v);(i), (ii), (iii), and (vi);(i), (ii), (iii), and (vi);(i), (ii), (iv), and (v);(i), (ii), (iv), and (vi);( i), (ii), (v), and (vi);(i), (iii), (iv), and (v);(i), (iii), (iv), and (vi);(i), (iii), (v), and (vi);(i), (iv), (v), and (vi);(ii), (iii), (iv), and (v);(ii), (iii), (iv), and (vi);(ii), (iii), (v), and (vi);(ii), (iii), (v), and (vi);(ii), (iv), (v), and (vi);(iii), (iv), (v), and (vi);(i), (ii), (iii), (iv), and (v);(i), (ii), (iii), (iv), and (vi);(i), (ii), (iii), (v), and (vi);(i), (ii), (iv), (v), and (vi);(i), (iii), (iv), (v), and (vi);(ii), (iii), (iv), (v), and (vi);(i), (ii), (iii), (iv), (v), and (vi);(i), (ii), (iii), (iv), (v), and (vi);(i), (ii), (iii), (iv), (v), and (vi);

[0073] Methods for measuring MHC (or HLA) binding ability are known in the art. For example, in silico analysis may be used to predict the affinity with which a peptide antigen will bind to an MHC (or HLA) molecule. Mass spectrometry may be used to identify peptides eluted from MHC (or HLA) molecules immunoprecipitated from target cells. Various biochemical assays for determining peptide-MHC (or peptide-HLA) binding are also available.

[0074] antigen binding molecule The present invention provides antigen-binding molecules that are (i) encoded by germline diversity in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) that bind to a peptide antigen present on the surface of a target cell in the individual, and optionally not present on the surface of a non-target cell in the individual. Such antigen-binding molecules are included in the immunotherapeutic agents selected or administered in the methods and medical applications described herein.

[0075] The antigen-binding molecule may be, for example, a T cell receptor (TCR), a chimeric antigen receptor (CAR), an antibody, an antibody fragment, or a bispecific T cell engager (BiTE). The antibody fragment may include a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a single chain variable fragment (scFv), a scFv-Fc, or a single domain antibody. Preferably, the antigen-binding molecule is a TCR or a CAR.

[0076] The antigen-binding molecule binds to the peptide antigen. The antigen-binding molecule may bind to the peptide antigen, for example, when the peptide antigen is bound to the MHC molecule. That is, the antigen-binding molecule may bind to the MHC-peptide antigen complex. Preferably, the binding between the peptide antigen and the antigen-binding molecule is non-covalent. The binding may be mediated, for example, by electrostatic interactions, hydrogen bonds, van der Waals forces, and / or hydrophobic interactions. Methods for detecting the binding between a peptide and an antigen-binding molecule are well known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. Functional testing methods for antigen-binding molecules are also known in the art. For example, cells may be genetically engineered to express the antigen-binding molecule. For example, if the antigen-binding molecule is a TCR, the α and β chains of the TCR may be inserted into T cells, such as human T cells, using a lentiviral vector. The peptide antigen is then added to the culture containing the cells expressing the antigen-binding molecule. Cellular activation (such as T cell activation) can be measured by well-known techniques such as (1) ELISpot for IFNγ secretion or (2) flow cytometry followed by immunostaining for intracellular cytokines and / or surface markers of activation. Technique (1) may be performed, for example, after 18 hours of peptide simulation. Technique (2) may be performed, for example, after 6-8 hours of peptide simulation.

[0077] The antigen binding molecule may comprise an antigen binding domain expressed by an immune cell derived from a donor having a genome that does not include germline diversity. The immune cell may be, for example, a T cell or a NKT cell. The antigen binding domain may comprise all or a portion of a TCR alpha chain, such as, for example, a TCR alpha chain disclosed herein. The antigen binding domain may comprise all or a portion of a TCR beta chain, such as, for example, a TCR beta chain disclosed herein. The antigen binding domain may comprise all or a portion of a TCR alpha chain (such as, for example, a TCR alpha chain disclosed herein) and all or a portion of a TCR beta chain (such as, for example, a TCR beta chain disclosed herein).

[0078] The antigen-binding domain may comprise one or more of the complementarity determining regions (CDRs) from the alpha chain of the TCR. For example, the antigen-binding domain may comprise two or three of the complementarity determining regions (CDRs) from the alpha chain of the TCR. The antigen-binding domain may comprise one or more of the complementarity determining regions (CDRs) from the beta chain of the TCR. For example, the antigen-binding domain may comprise two or three of the CDRs from the beta chain of the TCR. The antigen-binding domain may comprise one or more of the CDRs from the alpha chain and one or more of the CDRs from the beta chain of the TCR. For example, the antigen-binding domain may comprise (i) one of the CDRs derived from the α chain and one of the CDRs derived from the β chain, (ii) one of the CDRs derived from the α chain and two of the CDRs derived from the β chain, (iii) one of the CDRs derived from the α chain and three of the CDRs derived from the β chain, (iv) two of the CDRs derived from the α chain and one of the CDRs derived from the β chain, (v) two of the CDRs derived from the α chain and two of the CDRs derived from the β chain, (vi) two of the CDRs derived from the α chain and three of the CDRs derived from the β chain, (vii) three of the CDRs derived from the α chain and one of the CDRs derived from the β chain, (viii) three of the CDRs derived from the α chain and two of the CDRs derived from the β chain, or (ix) three of the CDRs derived from the α chain and three of the CDRs derived from the β chain. The antigen-binding domain may comprise one or more of: (a) a CDR1 derived from an alpha chain, (b) a CDR2 derived from an alpha chain, (c) a CDR3 derived from an alpha chain, (d) a CDR1 derived from a beta chain, (e) a CDR2 derived from a beta chain, and (f) a CDR3 derived from a beta chain.For example, the antigen binding domain may be: (a);(b);(c);(d);(e);(f);(a) and (b);(a) and (c);(a) and (d);(a) and (e);(a) and (f);(b) and (c);(b) and (d);(b) and (e);(b) and (f);(c) and (d);(c) and (e);(c) and (f);(d) and (e);(d) and (f);(e) and (f);(a), (b), (c);(a), (b), (d);(a), (b), and (e);(a), (b), and and (f);(a), (c), and (d);(a), (c), and (e);(a), (c), and (f);(a), (d), and (e);(a), (d), and (f);(a), (e), and (f);(b), (c), and (d);(b), (c), and (e);(b), (c), and (f);(b), (d), and (e);(b), (d), and (f);(b), (e), and (f);(c), (d), and (e);(c), (d), and (f);(c), (e), and (f);(d), (e ), and (f);(a), (b), (c), and (d);(a), (b), (c), and (e);(a), (b), (c), and (f);(a), (b), (d), and (e);(a), (b), (d), and (f);(a), (b), (e), and (f);(a), (c), (d), and (e);(a), (c), (d), and (f);(a), (c), (e), and (f);(a), (d), (e), and (f);(b), (c), (d), and (e);(b), (c), (d), and ( and (f);(b),(c),(e),(f);(b),(d),(e), and(f);(c),(d),(e), and(f);(a),(b),(c),(d),and(e);(a),(b),(c),(d),and(f);(a),(b),(c),(e),and(f);(a),(b),(c),(e),and(f);(a),(b),(d),(e),and(f);(a),(c),(d),(e),and(f);(b),(c),(d),(e),and(f);(a),(b),(c),(d),(e),and(f). Preferably, the antigen binding domain comprises a CDR3 derived from an alpha chain of a TCR and a CDR3 derived from a beta chain of a TCR. In either case, the TCR may be, for example, any TCR disclosed herein.

[0079] Exemplary antigen binding domains are described in the Examples (see Table 5 in Examples 1 and 5). An antigen binding domain may, for example, comprise a CDR3 from any one or more of the exemplary antigen binding domains. An antigen binding domain may, for example, comprise a CDR3 from an exemplary TCR alpha chain and a CDR3 from an exemplary TCR beta chain. An antigen binding domain may, for example, comprise the CDR1, CDR2, and CDR3 of an exemplary TCR alpha chain and the CDR1, CDR2, and CDR3 of an exemplary TCR beta chain.

[0080] The antigen binding domain may, for example, include any one or more of the exemplified antigen binding domains. The antigen binding domain may, for example, include the exemplified TCR alpha chain and the exemplified TCR beta chain.

[0081] The antigen-binding domain can be, for example, from one or more of: (a) SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290. and / or (b) a CDR3 derived from one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292. An antigen-binding molecule can comprise, for example, (a) a CDR1, CDR2, and CDR3 sequence from one or more of SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290. and / or (b) CDR1, CDR2, and CDR3 from one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292.The antigen-binding molecule can be, for example, (a) one of SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290. and / or (b) one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292.

[0082] An antigen-binding domain may comprise, for example, a CDR3 derived from one or more of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22. For example, an antigen-binding domain may comprise (a) a CDR3 derived from each of SEQ ID NOs: 6 and 8; (b) a CDR3 derived from each of SEQ ID NOs: 10 and 14 or 12 and 14; (c) a CDR3 derived from each of SEQ ID NOs: 16 and 18; (d) a CDR3 derived from each of SEQ ID NOs: 10 and 14; or (e) a CDR3 derived from each of SEQ ID NOs: 20 and 22.

[0083] An antigen-binding domain may comprise, for example, CDR1, CDR2, and CDR3 derived from one or more of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22. For example, an antigen-binding domain may comprise (a) CDR1, CDR2, and CDR3 derived from each of SEQ ID NOs: 6 and 8; (b) CDR1, CDR2, and CDR3 derived from each of SEQ ID NOs: 10 and 14 or 12 and 14; (c) CDR1, CDR2, and CDR3 derived from each of SEQ ID NOs: 16 and 18; (d) CDR1, CDR2, and CDR3 derived from each of SEQ ID NOs: 10 and 14; or (e) CDR1, CDR2, and CDR3 derived from each of SEQ ID NOs: 20 and 22.

[0084] An antigen-binding domain may comprise a CDR3 encoded within one or more nucleic acid sequences comprising or consisting of, for example, SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 to 31. For example, an antigen-binding domain may comprise a CDR3 encoded within each of (a) a nucleic acid sequence comprising or consisting of SEQ ID NO: 5 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 7; (b) a nucleic acid sequence comprising or consisting of SEQ ID NO: 9, a nucleic acid sequence comprising or consisting of SEQ ID NO: 11, and a nucleic acid sequence comprising or consisting of SEQ ID NO: 13; (c) a nucleic acid sequence comprising or consisting of SEQ ID NO: 15 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 17; (d) a nucleic acid sequence comprising or consisting of SEQ ID NO: 9 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 13; or (e) a nucleic acid sequence comprising or consisting of SEQ ID NO: 19 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 21. The antigen-binding domain may, for example, comprise a CDR3 encoded within each of (a) the nucleic acid sequence comprising or consisting of SEQ ID NO:23 and the nucleic acid sequence comprising or consisting of SEQ ID NO:24; (b) the nucleic acid sequence comprising or consisting of SEQ ID NO:25 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27, or the nucleic acid sequence comprising or consisting of SEQ ID NO:26 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27; (c) the nucleic acid sequence comprising or consisting of SEQ ID NO:28 and the nucleic acid sequence comprising or consisting of SEQ ID NO:29; (d) the nucleic acid sequence comprising or consisting of SEQ ID NO:25 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27; or (e) the CDR3 encoded within each of the nucleic acid sequences comprising or consisting of SEQ ID NO:30 and the nucleic acid sequence comprising or consisting of SEQ ID NO:31.

[0085] An antigen-binding domain may comprise, for example, CDR1, CDR2, and CDR3 encoded within one or more nucleic acid sequences comprising or consisting of SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 to 31. For example, an antigen-binding domain may comprise CDR1, CDR2, and CDR3 encoded within each of (a) a nucleic acid sequence comprising or consisting of SEQ ID NO:5 and a nucleic acid sequence comprising or consisting of SEQ ID NO:7; (b) a nucleic acid sequence comprising or consisting of SEQ ID NO:9, a nucleic acid sequence comprising or consisting of SEQ ID NO:11, and a nucleic acid sequence comprising or consisting of SEQ ID NO:13; (c) a nucleic acid sequence comprising or consisting of SEQ ID NO:15 and a nucleic acid sequence comprising or consisting of SEQ ID NO:17; (d) a nucleic acid sequence comprising or consisting of SEQ ID NO:9 and a nucleic acid sequence comprising or consisting of SEQ ID NO:13; or (e) a nucleic acid sequence comprising or consisting of SEQ ID NO:19 and a nucleic acid sequence comprising or consisting of SEQ ID NO:21. The antigen-binding domain may comprise, for example, CDR1, CDR2, and CDR3 encoded within each of: (a) the nucleic acid sequence comprising or consisting of SEQ ID NO:23 and the nucleic acid sequence comprising or consisting of SEQ ID NO:24; (b) the nucleic acid sequence comprising or consisting of SEQ ID NO:25 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27, or the nucleic acid sequence comprising or consisting of SEQ ID NO:26 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27; (c) the nucleic acid sequence comprising or consisting of SEQ ID NO:28 and the nucleic acid sequence comprising or consisting of SEQ ID NO:29; (d) the nucleic acid sequence comprising or consisting of SEQ ID NO:25 and the nucleic acid sequence comprising or consisting of SEQ ID NO:27; or (e) the CDR1, CDR2, and CDR3 encoded within each of the nucleic acid sequences comprising or consisting of SEQ ID NO:30 and the nucleic acid sequence comprising or consisting of SEQ ID NO:31.

[0086] The antigen-binding domain may comprise, for example, one or more of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 22. For example, the antigen-binding domain may comprise (a) SEQ ID NOs: 6 and 8; (b) SEQ ID NOs: 10 and 14 or 12 and 14; (c) SEQ ID NOs: 16 and 18; (d) SEQ ID NOs: 10 and 14; or (e) SEQ ID NOs: 20 and 22. The antigen-binding domain may be encoded by one or more nucleic acid sequences including or consisting of, for example, SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23-31. For example, the antigen-binding domain may be encoded by (a) a nucleic acid sequence comprising or consisting of SEQ ID NO:5 and a nucleic acid sequence comprising or consisting of SEQ ID NO:7; (b) a nucleic acid sequence comprising or consisting of SEQ ID NO:9, a nucleic acid sequence comprising or consisting of SEQ ID NO:11, and a nucleic acid sequence comprising or consisting of SEQ ID NO:13; (c) a nucleic acid sequence comprising or consisting of SEQ ID NO:15 and a nucleic acid sequence comprising or consisting of SEQ ID NO:17; (d) a nucleic acid sequence comprising or consisting of SEQ ID NO:9 and a nucleic acid sequence comprising or consisting of SEQ ID NO:13; or (e) a nucleic acid sequence comprising or consisting of SEQ ID NO:19 and a nucleic acid sequence comprising or consisting of SEQ ID NO:21. Preferably, the antigen-binding domain is encoded by (a) a nucleic acid sequence comprising or consisting of SEQ ID NO:23 and a nucleic acid sequence comprising or consisting of SEQ ID NO:24; (b) a nucleic acid sequence comprising or consisting of SEQ ID NO:25 and a nucleic acid sequence comprising or consisting of SEQ ID NO:27, or a nucleic acid sequence comprising or consisting of SEQ ID NO:26 and a nucleic acid sequence comprising or consisting of SEQ ID NO:27; (c) a nucleic acid sequence comprising or consisting of SEQ ID NO:28 and a nucleic acid sequence comprising or consisting of SEQ ID NO:29; (d) a nucleic acid sequence comprising or consisting of SEQ ID NO:25 and a nucleic acid sequence comprising or consisting of SEQ ID NO:27; or (e) a nucleic acid sequence comprising or consisting of SEQ ID NO:30 and a nucleic acid sequence comprising or consisting of SEQ ID NO:31.

[0087] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. For example, the antigen binding domain may be any of the following SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 3 8, 249, 250, 251, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. The antigen binding domain may, for example, comprise a CDR3 from each of SEQ ID NOs: 206 and 207. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 208 and 209, or each of 208 and 210. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 211 and 212. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 213 and 214.The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 215 and 216, or each of SEQ ID NOs: 215 and 217. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 218 and 219, or each of SEQ ID NOs: 218 and 220. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 221 and 223, or each of SEQ ID NOs: 222 and 223. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 224 and 225. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 226 and 227. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 228 and 229. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 230 and 231. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 232 and 234, or 233 and 234. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 235 and 236. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 237 and 238. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 239 and 240. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 241 and 242. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 243 and 245 or each of SEQ ID NOs: 244 and 245. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 246 and 247. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 248 and 249. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 250 and 251. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 252 and 253. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 254 and 255. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 256 and 257. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 258 and 259.The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 260 and 262, or each of SEQ ID NOs: 261 and 262. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 263 and 264, or each of SEQ ID NOs: 263 and 265. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 266 and 267. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 268 and 269. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 270 and 271. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 272 and 273. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 274 and 275. The antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 276 and 277. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 278 and 279. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 280 and 281. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 282 and 284 or 283 and 284. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 285 and 286. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 287 and 289 or each of SEQ ID NOs: 288 and 289. An antigen binding domain may, for example, comprise a CDR3 derived from each of SEQ ID NOs: 290 and 291 or each of SEQ ID NOs: 290 and 292.

[0088] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 1, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. For example, the antigen binding domain may be any of the sequences set forth in SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 206 and 207. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 208 and 209, or each of 208 and 210. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 211 and 212.The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 213 and 214. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 215 and 216, or each of SEQ ID NOs: 215 and 217. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 218 and 219, or each of SEQ ID NOs: 218 and 220. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 221 and 223, or each of SEQ ID NOs: 222 and 223. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 224 and 225. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 226 and 227. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 228 and 229. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 230 and 231. The antigen binding domain may, for example, comprise SEQ ID NOs: 232 and 234 or 233 and 234. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 235 and 236. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 237 and 238. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 239 and 240. The antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 241 and 242. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 243 and 245, or each of SEQ ID NOs: 244 and 245. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 246 and 247. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 248 and 249.An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 250 and 251. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 252 and 253. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 254 and 255. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 256 and 257. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 258 and 259. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 260 and 262 or each of SEQ ID NOs: 261 and 262. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 263 and 264, or each of SEQ ID NOs: 263 and 265. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 266 and 267. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 268 and 269. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 270 and 271. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 272 and 273. The antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 274 and 275. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 276 and 277. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 278 and 279. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 280 and 281. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 282 and 284 or 283 and 284. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 285 and 286.An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 287 and 289, or each of SEQ ID NOs: 288 and 289. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 from each of SEQ ID NOs: 290 and 291, or each of SEQ ID NOs: 290 and 292.

[0089] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 34 0, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, or 379. The antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO:293 and a nucleic acid sequence comprising or consisting of SEQ ID NO:294. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO:296, respectively, or within a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO:297, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of 298 and a nucleic acid sequence comprising or consisting of SEQ ID NO:299, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of 300 and a nucleic acid sequence comprising or consisting of SEQ ID NO:301, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of 302 and a nucleic acid sequence comprising or consisting of SEQ ID NO:303, respectively, or within a nucleic acid sequence comprising or consisting of 302 and a nucleic acid sequence comprising or consisting of SEQ ID NO:304, respectively.An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 306, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 307, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 309, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 310, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 311, respectively, and a nucleic acid sequence comprising or consisting of SEQ ID NO: 312, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 313 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 314, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 315 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 316. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 317 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 318. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 319 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 320 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 322 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 323. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 324 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 325. The antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO:326 and a nucleic acid sequence comprising or consisting of SEQ ID NO:327.An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 328 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 329. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 330 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 331 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 333 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 334. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 335 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 336. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 337 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 338. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 339 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 340. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 341 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 342. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 343 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 344. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 345 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 346. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 347 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 348 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349.An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 351, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 352, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 353 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 354, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 355 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 356, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 357 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 358, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 359 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 360, respectively. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 361 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 362. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 363 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 364. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 365 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 366. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 367 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 368. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 369 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 370 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371.An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 372 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 373. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 374 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 375 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376. An antigen binding domain may, for example, comprise a CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 378, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 379.

[0090] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, or 379. The antigen binding domain can, for example, comprise CDR1, CDR2, and CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO:293 and a nucleic acid sequence comprising or consisting of SEQ ID NO:294. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO:296, respectively, or within a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO:297, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of 298 and a nucleic acid sequence comprising or consisting of SEQ ID NO:299, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of 300 and a nucleic acid sequence comprising or consisting of SEQ ID NO:301, respectively.An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 302 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 303, or CDR1, CDR2, and CDR3 encoded within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 302 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 304. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 306, or within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 307. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 309, or within each of the nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and the nucleic acid sequence comprising or consisting of SEQ ID NO: 310. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 311 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 312, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 313 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 314, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 315 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 316, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 317 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 318, respectively. The antigen binding domain may, for example, comprise the CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 319 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 320 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321, respectively.An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 322 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 323, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 324 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 325, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 326 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 327, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 328 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 329, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 330 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 331 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 333 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 334, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 335 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 336, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 337 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 338, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 339 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 340, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 341 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 342, respectively.An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 343 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 344, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 345 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 346, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 347 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 348 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349, respectively. An antigen-binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 351, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 352, respectively. An antigen-binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 353 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 354, respectively. An antigen-binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 355 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 356, respectively. An antigen-binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 357 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 358, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 359 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 360, respectively. An antigen binding domain may, for example, comprise a CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 361 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 362, respectively.An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 363 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 364, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 365 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 366, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 367 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 368, respectively. An antigen-binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within a nucleic acid sequence comprising or consisting of SEQ ID NO: 369 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371, respectively, or within a nucleic acid sequence comprising or consisting of SEQ ID NO: 370 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371, respectively. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 372 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 373. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 374 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 375 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376. An antigen binding domain may, for example, comprise CDR1, CDR2, and CDR3 encoded within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 378, or within each of a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 379.

[0091] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. For example, the antigen binding domain may be any of the following SEQ ID NOs: 206, 207, 208, 209, 210, 211, 210, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 3 27, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 289, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, and 292. The antigen binding domain may comprise, for example, SEQ ID NOs: 206 and 207. The antigen binding domain may comprise, for example, SEQ ID NOs: 208 and 209 or 208 and 210. An antigen binding domain may, for example, comprise SEQ ID NOs: 211 and 212. An antigen binding domain may, for example, comprise SEQ ID NOs: 213 and 214. An antigen binding domain may, for example, comprise SEQ ID NOs: 215 and 216 or 215 and 217. An antigen binding domain may, for example, comprise SEQ ID NOs: 218 and 219 or 218 and 220.An antigen binding domain may comprise, for example, SEQ ID NOs: 221 and 223 or 222 and 223. An antigen binding domain may comprise, for example, SEQ ID NOs: 224 and 225. An antigen binding domain may comprise, for example, SEQ ID NOs: 226 and 227. An antigen binding domain may comprise, for example, SEQ ID NOs: 228 and 229. An antigen binding domain may comprise, for example, SEQ ID NOs: 230 and 231. An antigen binding domain may comprise, for example, SEQ ID NOs: 232 and 234 or 233 and 234. An antigen binding domain may comprise, for example, SEQ ID NOs: 235 and 236. An antigen binding domain may comprise, for example, SEQ ID NOs: 237 and 238. An antigen binding domain may comprise, for example, SEQ ID NOs: 239 and 240. An antigen binding domain may comprise, for example, SEQ ID NOs: 241 and 242. An antigen binding domain may comprise, for example, SEQ ID NOs: 243 and 245 or 244 and 245. The antigen binding domain may comprise, for example, SEQ ID NOs: 246 and 247. The antigen binding domain may comprise, for example, SEQ ID NOs: 248 and 249. The antigen binding domain may comprise, for example, SEQ ID NOs: 250 and 251. The antigen binding domain may comprise, for example, SEQ ID NOs: 252 and 253. The antigen binding domain may comprise, for example, SEQ ID NOs: 254 and 255. The antigen binding domain may comprise, for example, SEQ ID NOs: 256 and 257. The antigen binding domain may comprise, for example, SEQ ID NOs: 258 and 259. The antigen binding domain may comprise, for example, SEQ ID NOs: 260 and 262 or 261 and 262. The antigen binding domain may comprise, for example, SEQ ID NOs: 263 and 264 or 263 and 265. The antigen binding domain may comprise, for example, SEQ ID NOs: 266 and 267. The antigen binding domain may comprise, for example, SEQ ID NOs: 268 and 269. The antigen binding domain may comprise, for example, SEQ ID NOs: 270 and 271. An antigen binding domain may, for example, comprise SEQ ID NOs: 272 and 273. An antigen binding domain may, for example, comprise SEQ ID NOs: 274 and 275. An antigen binding domain may, for example, comprise SEQ ID NOs: 276 and 277. An antigen binding domain may, for example, comprise SEQ ID NOs: 278 and 279. An antigen binding domain may, for example, comprise SEQ ID NOs: 280 and 281. An antigen binding domain may, for example, comprise SEQ ID NOs: 282 and 284 or 283 and 284.An antigen binding domain may, for example, comprise SEQ ID NOs: 285 and 286. An antigen binding domain may, for example, comprise SEQ ID NOs: 287 and 289 or 288 and 289. An antigen binding domain may, for example, comprise SEQ ID NOs: 290 and 291 or 290 and 292.

[0092] Antigen binding domains can be, for example, those set forth in SEQ ID NOs: 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 373, 374, 375, 376, 377, 378, or 379. The antigen binding domain may be encoded by one or more nucleic acid sequences comprising or consisting of SEQ ID NO:293 and SEQ ID NO:294, for example. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 296, or a nucleic acid sequence comprising or consisting of 295 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 297. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of 298 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 299. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of 300 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 301. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of 302 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 303, or a nucleic acid sequence comprising or consisting of 302 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 304. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 306, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 305 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 307.The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 309, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 308 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 310. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of 311 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 312. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 313 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 314. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 315 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 316. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 317 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 318. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 319 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321, a nucleic acid sequence comprising or consisting of SEQ ID NO: 320 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 321. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 322 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 323. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 324 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 325. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 326 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 327. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 328 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 329. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 330 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 331 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 332.The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 333 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 334. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 335 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 336. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 337 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 338. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 339 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 340. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 341 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 342. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 343 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 344. The antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 345 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 346. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 347 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 348 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 349. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 351, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 350 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 352. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 353 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 354. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 355 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 356.The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 357 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 358. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 359 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 360. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 361 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 362. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 363 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 364. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 365 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 366. The antigen binding domain may, for example, be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 367 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 368. An antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 369 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 370 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 371. An antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 372 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 373. An antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 374 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 375 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 376. An antigen binding domain may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 378, or a nucleic acid sequence comprising or consisting of SEQ ID NO: 377 and a nucleic acid sequence comprising or consisting of SEQ ID NO: 379.

[0093] The immune cell may be, for example, a B cell or a plasma cell. The antigen-binding domain may, for example, comprise all or a portion of a light chain derived from an antibody or a B cell receptor (BCR). The antigen-binding domain may comprise all or a portion of a heavy chain derived from an antibody or a BCR. The antigen-binding domain may comprise all or a portion of a light chain and all or a portion of a heavy chain derived from an antibody or a BCR. The antigen-binding domain may comprise three CDRs derived from a light chain of an antibody or a BCR. The antigen-binding domain may comprise three CDRs derived from a heavy chain of an antibody or a BCR. The antigen-binding domain may comprise three CDRs derived from a light chain and three CDRs derived from a heavy chain of an antibody or a BCR. The antigen-binding domain may comprise a variable region of an antibody or a BCR. The antigen-binding domain may comprise Fab or (Fab')2 derived from an antibody or a BCR.

[0094] The antigen-binding molecule may comprise variants of the antigen-binding domain expressed by immune cells derived from a donor with a genome that does not contain germline diversity. Antigen-binding domains are described in detail in the previous paragraph. Over the entire length of the amino acid sequence of the antigen-binding domain, the variants preferably have at least 50% identity to the sequence. More preferably, the variants have at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of the antigen-binding domain. The variants retain the antigen-binding ability of the antigen-binding domain.

[0095] The antigen binding molecule may comprise or consist of an antigen binding molecule expressed by an immune cell derived from a donor with a genome that does not contain germline diversity. The immune cell may be, for example, a T cell or a NKT cell. The antigen binding molecule may be a TCR. The immune cell may be a B cell or a plasma cell. The antigen binding molecule may be an antibody or a BCR.

[0096] The antigen-binding molecule may comprise a variant of the antigen-binding molecule expressed by an immune cell derived from a donor having a genome that does not contain germline diversity. Antigen-binding molecules are described in the previous paragraph. Over the entire length of the amino acid sequence of the antigen-binding molecule, the variant preferably has at least 50% identity to the sequence. More preferably, the variant has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of the antigen-binding molecule. The variant retains the antigen-binding ability of the antigen-binding molecule.

[0097] The variant may comprise the CDRs of an antigen-binding molecule expressed by an immune cell derived from a donor with a genome that does not contain germline diversity. For example, the variant may comprise three CDRs derived from the alpha chain of a TCR expressed by an immune cell. The variant may comprise three CDRs derived from the beta chain of a TCR expressed by an immune cell. The variant may comprise three CDRs derived from the alpha chain and three CDRs derived from the beta chain of a TCR expressed by an immune cell. The variant may comprise three CDRs derived from the light chain of an antibody or BCR expressed by an immune cell. The variant may comprise three CDRs derived from the heavy chain of an antibody or BCR expressed by an immune cell. The variant may comprise three CDRs derived from the light chain and three CDRs derived from the heavy chain of an antibody or BCR expressed by an immune cell.

[0098] Immunotherapy The immunotherapeutic agent comprises an antigen-binding molecule that is i) encoded by germline diversity in an individual, (ii) capable of binding to an MHC molecule, and (iii) that binds to a peptide antigen present on the surface of a target cell in the individual and, optionally, not present on the surface of a non-target cell in the individual. Antigen-binding molecules and peptide antigens are described in more detail above.

[0099] The immunotherapeutic agent may further comprise an immune cell expressing an antigen-binding molecule on its surface. The immune cell may be autologous to the individual. The immune cell may be allogeneic to the individual. The immune cell may be derived from a donor having a genome that does not contain germline diversity. Preferably, the donor is disease-free. The immune cell may be, for example, a T cell, a B cell, a NKT cell, a NK cell, a monocyte, or a macrophage. Preferably, the immune cell is a T cell. The T cell may be a CD4+ T cell or a CD8+ T cell. Preferably, the T cell is a CD4+ T cell.

[0100] The antigen-binding molecule expressed on the surface of the immune cells may be derived from the donor. For example, the immune cells may be T cells or NKT cells derived from the donor. The antigen-binding molecule expressed on the surface of the immune cells derived from the donor may be TCR derived from the donor. In this way, the immune cells derived from the donor can naturally have specificity for peptide antigens. To provide allogeneic immune cell transplantation, an immunotherapeutic agent may be administered to the individual. After administration, the peptide antigen on the target cells may bind to the antigen-binding molecule, activating the immune cells and generating an immune response against the target cells expressing the peptide antigen. Thus, the disease in the individual can be treated.

[0101] The antigen-binding molecule expressed on the immune cell surface may be a T cell receptor (TCR) or a chimeric antigen receptor (CAR). By expressing a TCR or CAR specific for a peptide antigen, specificity for the peptide antigen can be imparted to immune cells that are autologous or allogeneic to the individual and would not otherwise recognize the peptide antigen. In this way, immune cells specific for the peptide antigen can be genetically engineered. Preferably, the immune cells are T cells. Preferably, the T cells are CD4+ T cells. After administration of the genetically engineered immune cells to an individual, the peptide antigen on the target cell can bind to the CAR, activating the immune cell and generating an immune response against the target cell expressing the peptide antigen. Thus, the disease in the individual can be treated.

[0102] The immunotherapeutic agent may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, and transdermal.

[0103] The immunotherapeutic agents are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The amount administered will depend on the individual being treated, the disease being treated, and the capacity of the subject's immune system. Precise amounts of immunotherapeutic agent required to be administered may depend on the judgment of the practitioner and may be peculiar to each individual.

[0104] The immunotherapeutic agent may include a pharma- ceutically acceptable carrier or diluent. Thus, the immunotherapeutic agent may be provided as a pharmaceutical composition. Typically, such compositions are prepared as a liquid suspension of the antigen-binding molecules and / or cells. The antigen-binding molecules and / or cells may be mixed with excipients that are pharma- ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, glucose, glycerol, and the like, and combinations thereof. The pharmaceutical composition may be formulated using any suitable method. Conventional methods in the pharmaceutical art may be used to formulate the antigen-binding molecules and / or cells with standard pharma- ceutical acceptable carriers and / or excipients. The exact nature of the formulation will depend on several factors, including the cells to be administered and the desired route of administration. Suitable types of formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA. In addition, if necessary, the pharmaceutical composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents and / or pH buffering agents.

[0105] Polynucleotides The present invention further provides a polynucleotide encoding the antigen-binding molecule or peptide antigen of the present invention. That is, the present invention provides a polynucleotide encoding an antigen-binding molecule that is (i) encoded by germline diversity in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. The present invention also provides a polynucleotide encoding a peptide antigen that is (i) encoded by germline diversity in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual. Any of the aspects described above in relation to the antigen-binding molecule or peptide of the present invention may also be applied to the polynucleotide of the present invention.

[0106] Polynucleotides may include RNA. Polynucleotides may include DNA. Polynucleotides may include RNA and DNA.

[0107] The polynucleotide may be, for example, any of the sequences set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329 , 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, or 379.

[0108] vector The present invention provides a vector comprising a polynucleotide of the present invention. That is, the present invention provides a vector comprising a polynucleotide encoding an antigen-binding molecule that is i) encoded by germline diversity in an individual, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally binds to a peptide antigen that is not present on the surface of non-target cells in the individual. The present invention also provides a vector comprising a polynucleotide encoding a peptide antigen that is (i) encoded by germline diversity in an individual with a disease, (ii) capable of binding to an MHC molecule, and (iii) present on the surface of target cells in the individual, and optionally not present on the surface of non-target cells in the individual.

[0109] The vector may be a viral vector. The viral vector may be, for example, a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, or a herpes simplex virus. Methods for producing and purifying such vectors are known in the art. The retrovirus may be a gamma retrovirus. The lentivirus may be a modified HIV virus suitable for use in gene delivery. The lentivirus may be an SIV, FIV, or Equine Infectious Anemia Virus (EQIA) based vector. The viral vector may include a targeting molecule to ensure efficient transduction with the nucleic acid sequence or nucleic acid construct. The targeting molecule is usually provided entirely or partially on the surface of the viral vector to enable the molecule to target the virus to the cell. The viral vector is preferably replication-deficient.

[0110] The vector may be a non-viral vector. Preferably, the non-viral vector is a DNA plasmid, a naked nucleic acid, a nucleic acid complexed with a delivery vehicle, or an artificial virion. The non-viral vector may be a human artificial chromosome, for example, as described in Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). When the non-viral vector is a nucleic acid complexed with a delivery vehicle, the delivery vehicle may be a liposome, a virosome, or an immunoliposome. The integration of the plasmid vector may be facilitated by a transposase such as sleeping beauty or PiggyBAC. EXAMPLES

[0111] Example 1 - Clarifying the antigenic basis of GvL Given the low mutational burden in AML, HLA-linked GvL antigens are likely due to mismatched expression of germline variants (i.e., non-major histocompatibility antigens) between patient and donor rather than neoantigens. It remains unclear whether donor GvL responses are driven by public antigens (those with a population frequency of >0.05) or by less frequent private antigens.

[0112] We performed an unbiased approach to identify GvL antigens in bone marrow samples from 15 AML patients (12 of 15 were cured, 3 of 15 died of relapse) treated with allo-SCT. First, we performed whole exome sequencing (WES) of patient and donor DNA to identify coding variants that were mismatched between the patient and donor, and then performed RNA-Seq to identify all variants that were expressed in the patient's AML cells and were mismatched between the patient's AML cells and the donor. As expected, more germline mismatched variants were identified in unrelated donor transplants compared to sibling donor transplants (5744 vs. 3253 variants, Figure 1a and b).

[0113] Of all variants, 98.7% were germline variants, with only 1.3% being AML-specific somatic variants. An average of 28.6% of variants were detectable in the RNA-Seq reads. Of all variants, 47.8% were present in more than one patient. Variants present in multiple patients are likely to be important for clinical translation. From these variants, we identified putative GvL antigens using two methods: 1. In silico prediction of HLA binding affinity using exome-identified variants and RNA-Seq expression data derived from AML. 2. Mass spectrometry of peptides presented by HLA classes I and II.

[0114] First, putative GvL antigens were identified by integrating WES-identified variants, RNA-Seq expression data, and patient-specific HLA alleles. Then, in silico HLA class I and II binding predictions were performed using NetMHCpan 4.0 and NetMHCIIpan 3.0, utilizing artificial neural networks trained on HLA binding data. For HLA classes I and II, 150–700 mismatched HLA-binding epitopes were predicted per patient (Figure 2). On average, 2864 genes were contained in the potential immunogenic variants / patient likelihood. By RNA-Seq, 56% of these genes were expressed in AML blasts.

[0115] In the second approach, HLA classes I and II were immunoprecipitated from AML blasts, and HLA-binding peptides were eluted and analyzed by mass spectrometry. Patient-specific proteomic databases were generated using variants identified in the exome. Peptide sequences were then identified from mass spectra by referencing these databases. Immunopeptidomic analysis revealed 2.1 patient-donor mismatched peptides per patient (compared to 150-700 mismatched peptides by computational approaches). This is not surprising, since immunopeptidomic analysis identifies only a subset of the highest affinity and most ionizable HLA-binding peptides. Nevertheless, some predicted mismatched peptides were confirmed.

[0116] Characterization of GvL T cell responses For any individual patient, it is unclear whether the GvL response is mediated by a few dominant T cell clones or whether multiple clones contribute to the anti-AML response. To begin to address this, we established a general workflow. As proof of principle, we investigated one patient. Using a sensitive cultured IFNγ ELISpot assay, a library of 335 mismatched peptides identified by in silico prediction and mass spectrometry was screened for their ability to activate donor-derived T cells from a patient after allo-SCT. The patient had a strong T cell response to two overlapping peptides (SEQ ID NOs: 3 and 4, see below) targeting the same germline mismatched variant.

[0117] The single nucleotide polymorphism (SNP) that produced the immunogenic peptide is a C→T substitution in PADI4 (peptidylarginine deiminase 4), resulting in a serine (S)→phenylalanine (F) amino acid substitution. (dbSNP ID=rs1748020). Expression of PADI14 in normal human tissues, human tumors, and primary AML samples is shown in Figure 5. In an initial IFNγ ELISpot screen, two peptides overlapping with this PADI4 SNP elicited T cell responses: Peptide 1 (SEQ ID NO:1) LTISLLDTFNLELPEAVVFQ Peptide 2 (SEQ ID NO:2) ISLLDTFNLELPEAVVFQDS

[0118] Subsequent ELISpot experiments were used to identify nested peptides within these longer sequences that elicited the strongest T cell responses: Nested peptide 1 (SEQ ID NO:3) SLLDTFNLELPEAVVF Nested peptide 2 (SEQ ID NO:4) LDTFNLELPEAVVFQ

[0119] Using IFNγ ELISpot with HLA-blocking antibodies, we demonstrated that allo-AML responses were restricted to HLA-DP (Figure 3a). Supporting this finding, immunophenotypic analysis of peptide-responsive T cells, identified by cytokine (IFNγ, TNFα) production and expression of a degranulation marker (CD107a), revealed that the response was mediated by CD4+ but not CD8+ T cells (Figure 3b). IFNγ ELISpot with HLA-blocking antibodies demonstrated that SEQ ID NOs: 1-4 all induced T cell responses that were restricted to HLA-DP.

[0120] Consistent with our findings, the importance of HLA class II is supported by recent evidence that HLA class II expression is downregulated in AML blasts after allo-SCT relapse in one-third of patients, i.e., it is a common mechanism of immune evasion. Of note, this germline polymorphism is present in 8% of the Caucasian population, and the DP allele is present in approximately 60% of the Caucasian population, suggesting that it may be a broadly applicable target GvL antigen.

[0121] The following protocol was used to determine the sequence of the TCR recognizing peptide 1 (SEQ ID NO:1) or peptide 2 (SEQ ID NO:2): 1. Post-transplant blood is cultured with peptides (identified from IFNγ ELISpot screening) to enrich for antigen-specific T cells. 2. After peptide stimulation, activated (IFNγ+) and control (IFNγ-) T cells are sorted by FACS. 3. Single-cell RNA sequencing with V(D)J enrichment.

[0122] As demonstrated in Figure 6, single cell sequencing identified two expanded clones (clone 1 and clone 2) within the CD4+IFNγ+ population. The two expanded clones are putative antigen-specific T cells. As shown in Figure 7, the putative antigen-specific T cells express multiple cytokines (IFNγ, TNFα) and activation markers (FASLG, IL2RA, TNFRSF9, GZMB) in response to peptide stimulation.

[0123] Single-cell RNA sequencing was further used to determine the sequences of TCRs recognizing nested peptide 1 (SEQ ID NO: 3) or nested peptide 2 (SEQ ID NO: 4). SEQ ID NOs: 5-22 provide the α- and β-chain sequences of the five most frequent T cell clones in the experiment enriching for antigen-specific T cells. Clone 2 contains two α-sequences and one β-sequence. Clone 4 has one of these α-chains and the same β-chain, demonstrating that clones 2 and 4 very likely recognize the same epitope.

[0124] Clone 1-TCRα Nucleotide sequence (SEQ ID NO:5) JPEG2024514705000004.jpg118170 (the consensus sequence ends before the end of TRAC) Amino acid sequence (SEQ ID NO:6) JPEG2024514705000005.jpg36170 (the consensus sequence ends before the end of TRAC) No underline = 5'UTR JPEG2024514705000006.jpg3049 Lower case = CDR3 (17aa length)

[0125] Clone 1-TCRβ Nucleotide consensus sequence (SEQ ID NO:7) JPEG2024514705000007.jpg125170 (the consensus sequence ends before the end of TRBC2) Amino acid consensus sequence (SEQ ID NO:8) JPEG2024514705000008.jpg45170 (the consensus sequence ends before the end of TRBC2) No underline = 5'UTR JPEG2024514705000009.jpg3453 lower case = CDR3 (16aa length)

[0126] Clone 2-TCRα(1) Nucleotide consensus sequence (SEQ ID NO:9) JPEG2024514705000010.jpg113170 (the consensus sequence ends before the end of TRAC) Amino acid consensus sequence (SEQ ID NO: 10) JPEG2024514705000011.jpg38170 (the consensus sequence ends before the end of TRAC) No underline = 5'UTR JPEG2024514705000012.jpg3257 Lower case = CDR3 (13aa length)

[0127] Clone 2-TCRα(2) Nucleotide consensus sequence (SEQ ID NO:11) JPEG2024514705000013.jpg114170 (the consensus sequence ends before the end of TRAC) Amino acid consensus sequence (SEQ ID NO: 12) JPEG2024514705000014.jpg38170 (the consensus sequence ends before the end of TRAC) No underline = 5'UTR JPEG2024514705000015.jpg3350 lowercase = CDR3 (12aa length)

[0128] Clone 2-TCRβ Nucleotide consensus sequence (SEQ ID NO: 13) JPEG2024514705000016.jpg142170 (the consensus sequence ends before the end of TRBC1) Amino acid consensus sequence (SEQ ID NO: 14) JPEG2024514705000017.jpg38170 (the consensus sequence ends before the end of TRBC1) No underline = 5'UTR JPEG2024514705000018.jpg4666 lowercase = CDR3 (14aa length)

[0129] Clone 3-TCRα Nucleotide consensus sequence (SEQ ID NO: 15) JPEG2024514705000019.jpg113170 (the consensus sequence ends before the end of TRAC) Amino acid consensus sequence (SEQ ID NO: 16) JPEG2024514705000020.jpg36170 (the consensus sequence ends before the end of TRAC) No underline = 5'UTR JPEG2024514705000021.jpg3251 lowercase = CDR3 (12aa length)

[0130] Clone 3-TCRβ Nucleotide consensus sequence (SEQ ID NO: 17) JPEG2024514705000022.jpg107170 (the consensus sequence ends before the end of TRBC2) Amino acid consensus sequence (SEQ ID NO: 18) JPEG2024514705000023.jpg37170 (the consensus sequence ends before the end of TRBC2) No underline = 5'UTR JPEG2024514705000024.jpg4454 lower case = CDR3 (19aa length)

[0131] Clone 4 TCRα(1) and TCRβ derived from clone 2

[0132] Clone 5-TCRα Nucleotide consensus sequence (SEQ ID NO: 19) JPEG2024514705000025.jpg142170 (the consensus sequence ends before the end of TRAC) (Consensus and reference 5'UTR sequences differ - references shown) Amino acid consensus sequence (SEQ ID NO:20) JPEG2024514705000026.jpg38170 (the consensus sequence ends before the end of TRAC) No underline = 5'UTR JPEG2024514705000027.jpg3160 lowercase = CDR3 (14aa length)

[0133] Clone 5-TCRβ Nucleotide consensus sequence (SEQ ID NO:21) JPEG2024514705000028.jpg107170 (the consensus sequence ends before the end of TRBC2) Amino acid consensus sequence (SEQ ID NO:22) JPEG2024514705000029.jpg37170 (the consensus sequence ends before the end of TRBC2) No underline = 5'UTR JPEG2024514705000030.jpg4453 lowercase = CDR3 (13aa length)

[0134] SEQ ID NOs: 23-31 provide human codon-optimized versions of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, and 21, respectively. These human codon-optimized sequences encode the same amino acid sequences as SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, and 21, but can be more easily expressed in human cells than the original nucleotide sequences. Thus, the human codon-optimized sequences can be used in therapeutic applications in preference to the original nucleotide sequences. For each of SEQ ID NOs: 23-31: JPEG2024514705000031.jpg4370

[0135] Clone 1-TCRα (SEQ ID NO:23) JPEG2024514705000032.jpg72170

[0136] Clone 1-TCRβ (SEQ ID NO:24) JPEG2024514705000033.jpg84170

[0137] Clone 2-TCR alpha (1) (SEQ ID NO: 25) JPEG2024514705000034.jpg66170

[0138] Clone 2-TCR alpha (2) (SEQ ID NO: 26) JPEG2024514705000035.jpg67170

[0139] Clone 2-TCRβ (SEQ ID NO:27) JPEG2024514705000036.jpg84170

[0140] Clone 3-TCRα (SEQ ID NO:28) JPEG2024514705000037.jpg67170

[0141] Clone 3-TCRβ (SEQ ID NO:29) JPEG2024514705000038.jpg83170

[0142] Clone 5-TCRα (SEQ ID NO:30) JPEG2024514705000039.jpg73170

[0143] Clone 5-TCRβ (SEQ ID NO:31) JPEG2024514705000040.jpg83170

[0144] conclusion These data provide evidence that this is the first group to successfully implement an unbiased approach of the type employed here to identify GvL antigens, and that we have also identified the cognate TCR that recognizes the antigen, opening new ground in immunology and development of antigen-binding molecule-based therapeutics, including TCR-based therapeutics, as well as peptide vaccination with the identified antigen.

[0145] Example 2 - Further clarifying the antigenic basis of GvL Using the general workflow established in Example 1, the antigenic basis of GvL in further patients was elucidated. Briefly, mismatched peptides identified by in silico prediction and mass spectrometry for each patient were screened for their ability to activate donor-derived T cells taken from patients after allo-SCT using IFNγ ELISpot assay. HLA restriction was investigated using IFNγ ELISpot with HLA-blocking antibodies. Post-transplant blood was cultured with peptides for 12 days and then stimulated with peptides to evaluate the nature and frequency of peptide-responsive T cells (i.e., cells that produce cytokines (IFNγ, TNFα) and express degranulation markers (CD107a) after contact with peptides). Responses were evaluated by flow cytometry.

[0146] The results are shown in Table 3.

[0147] The mismatched peptide sequences expressed by the patient are shown in column D of Table 3. Amino acids that were mismatched between the patient and donor are underlined. In many cases, there are two peptides covering the same genetic variant that both elicit a T cell response.

[0148] Genetic variants causing patient-donor discrepancies are listed in columns F / G / H / I. dbSNP ID (column I) points to online databases of inherited genetic variants previously described.

[0149] Column J indicates whether the T cell response is against the variant or reference amino acid sequence. The reference amino acid sequence used was NCBI Human Reference Genome Build hg38, also known as GRCh38 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000001405.26). In most cases, the donor is homozygous for the reference allele and the patient is heterozygous, so the T cell response is against the variant sequence. However, in some cases, especially for common genetic variants, the response is against the reference sequence. In these cases, the donor is homozygous for the variant allele and the patient is either homozygous reference or heterozygous.

[0150] Column K indicates the allele frequency of the variant in the population and is taken from the gnomAD database.

[0151] The L / M / N / O column indicates the results of HLA restriction and immunophenotyping experiments.

[0152] [Table 3-1]

[0153] [Table 3-2]

[0154] [Table 3-3]

[0155] [Table 3-4]

[0156] [Table 3-5]

[0157] [Table 3-6]

[0158] Example 3 - Exome-seq, RNA-seq, and peptide screening by IFNγ ELISpot identify putative GvL antigens in 50 patient-donor pairs To identify common GvL antigens across a larger patient cohort and expand the repertoire of putative GvL antigens, the workflow described above (exome sequencing, RNA-Seq, and in silico antigen prediction) will be applied at scale to biobanked AMADEUS clinical trial samples. In addition to identifying multiple antigen targets, HLA restriction will be specified in each case.

[0159] AMADEUS offers a unique opportunity to obtain sample and clinical metadata (Figure 4). Recruitment began in June 2019 and will be carried out in all UK allo-SCT transplant centres. The recruitment target is 324 patients over 3 years (recruiting 108 patients per year). 49 patients were recruited by March 2020 (ahead of recruitment schedule). Recruitment stopped in March (COVID) and resumed in May. Predicted relapses: year 1 - 21, year 2 - 49, year 3 - 59, year 4 - 24, year 5 - 10 patients.

[0160] Exome sequencing will be performed on 50 patient-donor pairs. For patients, AML cells will be used for sequencing and T cells will serve as germline controls. For donors, DNA will be available from the NHS tissue typing centre. RNA sequencing of AML cells from each patient will also be performed. Sequence data will be analysed to identify patient-donor mismatched peptides that are predicted to be expressed on AML cells and bind to patient-specific HLA molecules. These peptides will be screened for their ability to activate donor-derived T cells from post-transplant blood samples using ex vivo and highly sensitive cultured IFNγ ELISpot assays. Component peptides of pools that elicit ELISpot responses will be tested individually to identify the recognised peptide(s) and T cell phenotype (CD4 or CD8) and determine the HLA restriction of responding T cells by flow cytometric analysis and antibody blocking approaches (Figure 3).

[0161] Patient selection is based on identifying patients most likely to have initiated a GvL response. Patient selection excludes: (i) patients unlikely to have developed GvL, (ii) patients with early relapse within 3 months after allo-SCT, as these patients will undergo allo-SCT with recalcitrant disease and relapse before adaptive GvL T cell responses can control the disease. Patient selection includes: (i) patients who have received CC-486 (approximately 40 patients) and those who have not as controls (approximately 10 patients), (ii) patients who received allo-SCT with low levels of disease (known as measurable residual disease or MRD) that then gradually disappeared after allo-SCT, as GvL may have cleared the disease, and (iii) patients who lost detectable MRD and subsequently relapsed (after 12 months), as this may help elucidate the mechanism of GvL loss.

[0162] GvL antigens have been shown to be germline polymorphisms that can be targeted not only in AML but also in multiple cancers. Given that peptide binding to HLA class II is often promiscuous, some peptides bind to multiple HLA alleles.

[0163] Ultimately, we hope to develop "off the shelf" engineered T cell therapies with suitable combinations to treat the majority of patients based on gene polymorphism and HLA type.

[0164] Example 4 - Detailed characterization of GvL T cell responses and evaluation of the role of CC-486 (oral azacytidine) in enhancing immune surveillance and disease management There are multiple mechanisms by which AML may evade GvL, including: reduced HLA presentation, increased expression of immune checkpoint molecules by T cells and their ligands on AML blasts, expression of immunosuppressive molecules such as indoleamine 2,3-dioxygenase 1 (IDO1), and altered pro- and anti-inflammatory cytokine milieu. Treatment with the DNA methyltransferase inhibitor azacytidine (aza) after transplantation had beneficial clinical effects, but the mechanism is unclear. On the other hand, aza can promote GvL, upregulating the expression of melanoma-associated antigens (MAGEs) and triggering CD8+ T cell responses. Alternatively, aza may exert a direct anti-leukemic effect to buy time for a therapeutic GvL response to occur.

[0165] To establish how CC-486 works, patients in the Amadeus trial were studied in detail as follows:

[0166] i. Longitudinal tracking of antigen-specific anti-AML T cell responses Flow cytometry-based methods are used to identify T cells responding to antigenic peptides based on upregulation of activation markers, production of cytokines (IFNγ / TNFα) or upregulation of degranulation markers (CD107a) after peptide stimulation, and / or using fluorescently labeled HLA-peptide tetramers (mapped with IFNγ ELISpot assays). A multiparameter flow cytometry panel (read out on a Cytek Aurora Spectral flow cytometer or BD Symphony, which readily allows for the simultaneous analysis of at least 30 parameters) will be employed to allow assessment of naive, memory, effector, and regulatory T cells, as well as maturation / differentiation markers (CD45RA / RO, CCR7, CD27, CD28, CD57); lineage-specific transcription factors (Tbet, Eomes, GATA3, RORγt, FoxP3); cytolytic effector functions (perforin, granzymes A and B, Fas, FasL, TRAIL); and inhibitory receptors that are often co-expressed on exhausted T cells (PD-1, CTLA-4, LAG3, TIM3, TIGIT). NK cells, which may have both effector and immunoregulatory roles, will also be examined.

[0167] To track the clonal dynamics of GvL T cells and determine whether CC-486 promotes, maintains, and amplifies GvL T cell responses, multiparameter flow cytometry-based analysis of antigen-specific T cell responses will be performed on longitudinal serial samples from each patient. This will allow us to evaluate (i) the time after transplant when maximal GvL clonal expansion occurs, (ii) the correlation between GvL-specific T cell frequency and immunophenotype, the amount of disease and clinical outcome, CC-486 treatment, GvHD, and the amount of immunosuppression, and (iii) the status of GvL-specific T cells at the time of relapse. In addition to providing correlative data to suggest possible biological mechanisms of disease control by CC-486, we can also provide prognostic biomarker data. Loss of antigen-specific T cell clones will be evaluated by comparing patients who relapsed after allo-SCT with those who did not, and whether these T cell clones display an exhausted phenotype.

[0168] ii. Assess how GvL responses shape the T cell repertoire after transplantation Bulk and single cell (sc) T cell receptor (TCR) sequencing of donor-derived AML-reactive T cells in patient blood and bone marrow samples will be performed, which can be combined with whole transcriptome sequencing to further define the transcriptional status of antigen-specific T cell clones.

[0169] In a pilot experiment, we successfully stimulated PBMCs from one patient with overlapping putative GvL peptides identified by IFNγ ELISpot mapping (see Figure 3) or control peptides, and subjected responding T cells (identified by IFNγ secretion using capture reagents) to FACS sorting and parallel single-cell TCR / transcriptome sequencing (10× genomics platform) (Keskin, DB et al. Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial. Nature, 2019. 565(7738):p.234-239). Analysis of the data allowed us to identify TCRs highly enriched in the peptide-responsive population and to simultaneously investigate their gene expression profile. To allow us to determine the frequency of each peptide-responsive TCR in the in vivo T cell repertoire, we also performed bulk TCR sequencing on unstimulated ex vivo samples from the same patient. This established a workflow in our laboratory that can be easily applied to the large sample set of the AMADEUS trial.

[0170] Single cell sequencing enables: (i) Identify the paired TCR α and β chains of GvL T cell clones. (ii) characterize the phenotype of GvL T cells from the transcriptome; (iii) Assess the size of antigen-specific T cell clones ex vivo and after stimulation.

[0171] iii. Antigen-specific T cell-mediated killing of AML cells Peptide-specific T cell clones are generated using a single-cell cloning method established by the inventors (Brenna, E. et al. CD4(+)T Follicular Helper Cells in Human Tonsils and Blood Are Clonally Convergent but Divergent from Non-Tfh CD4(+)Cells. Cell Rep, 2020.30(1):p.137-152 e) and / or donor-derived CD4 T cells are transduced with lentiviral vectors encoding paired TCR α and β chains to generate antigen-specific T cell populations. Using flow cytometry-based methods, these are tested for in vivo activation (upregulation of CD69, CD25, and CD137), cytokine (IFNγ and TNFα) production, degranulation (expression of surface CD107a), and cytotoxicity (assessed by loss of fluorescent dye-labeled target cells) after incubation with patient AML cells loaded with the relevant peptide (and / or an irrelevant peptide as control). Patients with the most polymorphic antigens and the most common HLA types are selected. Priority is given to those with the largest T cell clones.

[0172] Xenograft models derived from AML patients are created in immunodeficient NSG / NSGW mice. In engraftment samples after AML establishment, the survival rates of mice and the percentage of AML cells were compared after injecting T cell clones or patient T cells transduced with either TCRs that recognize the GvL antigen derived from the AML or TCRs that recognize a control peptide. In this way, it can be confirmed that TCR-transduced T cells kill AML in vivo in the AML patients used.

[0173] iv. Quantitate remaining AML cells in bone marrow samples Highly sensitive flow cytometry and next generation sequencing are used to precisely quantify remaining AML cells after treatment, called measurable residual disease analysis (MRD), at two time points: before enrollment in the trial and 3 months after allo-SCT. The sensitivity of this approach is as high as 10 cells.5 There is one AML cell in each sample.

[0174] v. Assess HLA expression in AML blasts To investigate loss / reduction of HLA expression as a mechanism of immune evasion, RNA-Seq performed on AML cells at diagnosis will be performed at relapse after transplantation. Changes in expression of transcriptional and signaling regulators of HLA expression (e.g., CIITA, RFX5, NFYA, NFYB, IL-1A, IL-1B, IRF8), genes controlling peptide processing, and ligands of inhibitory receptors and immunosuppressants expressed on T cells will be evaluated. Expression of HLA proteins on the cell surface will also be represented by flow cytometry. This will allow us to study the role of CC-486 in regulating different modes of GvL failure.

[0175] Example 5 The following protocol was used to sequence the TCRs that recognize the cognate peptide antigens listed in Table 3 above: 1. Thaw post-transplant PBMCs from patients in whom peptide antigens were detected. Culture the cells with peptide, IL-2, and IL-7 for 12 days. 2. Allow the cells to rest overnight. 3. Stimulate the cells with peptide for 6 hours. 4. Stain cells for FACS with antibodies and IFNγ capture reagents. 5. Perform FACS sorting of activated (IFNγ+) CD4+ or CD8+ T cells and negative control (IFNγ-) populations. 6. Perform 10X Chromium single-cell RNA sequencing with V(D)J enrichment.

[0176] T cells recognizing 18 of the 24 peptides shown in Table 3 (including PADI4) were detectable by FACS. Of these, 17 were detectable using IFNγ capture. The data are shown in Tables 4 and 5 below and in Figures 8-23. Table 5 includes clones that were (a) larger in size than the largest clone in the control repertoire or (b) >10% of the repertoire, even if they were not larger than the largest clone in the control repertoire.

[0177] [Table 4]

[0178] [Table 5-1]

[0179] [Table 5-2]

[0180] [Table 5-3]

[0181] [Table 5-4]

[0182] [Table 6-1]

[0183] [Table 6-2]

[0184] [Table 6-3]

[0185]

Table 6-4

[0186]

Table 6-5

[0187]

Table 6-6

[0188]

Table 6-7

[0189]

Table 6-8

[0190]

Table 6-9

[0191]

Table 6-10

[0192]

Table 6-11

[0193]

Table 6-12

[0194]

Table 6-13

[0195]

Table 6-14

[0196]

Table 6-15

[0197]

Table 6-16

[0198]

Table 6-17

[0199]

Table 6-18

[0200]

Table 6-19

[0201]

Table 6-20

[0202]

Table 6-21

[0203]

Table 6-22

[0204]

Table 6-23

[0205]

Table 6-24

[0206]

Table 6-25

[0207]

Table 6-26

[0208]

Table 6-27

[0209]

Table 6-28

[0210]

Table 6-29

[0211]

Table 6-30

[0212]

Table 6-31

[0213]

Table 6-32

[0214]

Table 6-33

[0215]

Table 6-34

[0216]

Table 6-35

[0217]

Table 6-36

Claims

1. An antigen-binding molecule that (i) is encoded by germline variation in an individual with a disease, (ii) is capable of binding to an MHC molecule, and (iii) binds to a peptide antigen that is present on the surface of a target cell in said individual, and optionally is not present on the surface of a non-target cell in said individual.

2. The antigen-binding molecule of claim 1 , wherein the MHC molecule is an MHC class I molecule or an MHC class II molecule.

3. The antigen-binding molecule of claim 1, wherein the peptide antigen binds to an antigen-binding domain expressed by an immune cell derived from a donor having a genome that does not contain the germline diversity.

4. The antigen-binding molecule of claim 3, wherein the donor is disease-free.

5. The antigen-binding molecule of claim 3, wherein the germline diversity comprises one or more nucleotide substitutions, insertions, and / or deletions relative to the donor genome.

6. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule is a T cell receptor (TCR), a chimeric antigen receptor (CAR), an antibody, an antibody fragment, or a bispecific T cell engager (BiTE).

7. The antigen-binding molecule of claim 6, wherein the antibody fragment comprises a fragment antigen-binding (Fab) fragment, an F(ab')2 fragment, a single-chain variable fragment (scFv), an scFv-Fc, or a single domain antibody.

8. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises an antigen-binding domain or a variant thereof expressed by an immune cell derived from a donor having a genome that does not contain the germline diversity.

9. An immunotherapeutic agent comprising an antigen-binding molecule according to any one of claims 1 to 8.

10. The immunotherapeutic agent of claim 9, further comprising an immune cell expressing an antigen-binding molecule on its surface, optionally wherein the immune cell is a T cell.

11. The immunotherapy of claim 10, wherein the immune cells are derived from a donor having a genome that does not contain the germline diversity.

12. The immunotherapeutic agent of claim 10 , wherein the donor is disease-free.

13. The immunotherapeutic agent of claim 10 , wherein the antigen-binding molecule is a TCR.

14. The immunotherapeutic agent of claim 10, wherein the antigen-binding molecule is a CAR, and optionally the immune cells are autologous or allogeneic to the individual.

15. The immunotherapeutic agent of claim 9 for use in a method of treating a disease in an individual, the method comprising administering the immunotherapeutic agent to the individual.

16. The antigen-binding molecule of claim 1, wherein the disease is cancer and the target cell is a cancer cell.

17. The immunotherapy agent described in claim 9, wherein the disease is cancer and the target cells are cancer cells.

18. The antigen-binding molecule described in claim 16, wherein the cancer is acute myeloid leukemia (AML) and the cancer cells are AML cells.

19. The immunotherapy agent described in claim 17, wherein the cancer is acute myeloid leukemia (AML) and the cancer cells are AML cells.

20. A polynucleotide encoding the antigen-binding molecule of any one of claims 1 to 8.

21. A vector comprising the polynucleotide of claim 20.

22. The antigen-binding molecule of claim 1, wherein the peptide antigen comprises or consists of one or more of SEQ ID NOs: 392, 393, 395, 384, 385, 389, 1-4, 380-383, 386-388, 390, 391, 394, and 396-412.

23. The peptide antigen, (a) SEQ ID NO: 392 or 393; (b) SEQ ID NO: 395; (c) SEQ ID NO: 384 or 385; or (d) SEQ ID NO: 389 The antigen-binding molecule of claim 1, comprising or consisting of:

24. The antigen-binding molecule, (a) a CDR3 derived from one or more of SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290; and / or (b) a CDR3 derived from one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292. The antigen-binding molecule of claim 1 .

25. The antigen-binding molecule, (a) CDR1, CDR2, and CDR3 derived from one or more of SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290; and / or (b) CDR1, CDR2, and CDR3 from one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292. The antigen-binding molecule of claim 24 .

26. The antigen-binding molecule, (a) one or more of SEQ ID NOs: 6, 10, 12, 16, 20, 206, 208, 211, 213, 215, 218, 221, 222, 224, 226, 228, 230, 232, 233, 235, 237, 239, 241, 243, 244, 246, 248, 250, 252, 254, 256, 258, 260, 261, 263, 266, 268, 270, 272, 274, 276, 278, 280, 282, 283, 285, 287, 288, and 290; and / or (b) The antigen-binding molecule of claim 25, comprising one or more of SEQ ID NOs: 8, 14, 18, 22, 207, 209, 210, 212, 214, 216, 217, 219, 220, 223, 225, 227, 229, 231, 234, 236, 238, 240, 242, 245, 247, 249, 251, 253, 255, 257, 259, 262, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 284, 286, 289, 291, and 292.

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