EA042409B1 - CD3-BINDING POLYPEPTIDES - Google Patents

Description

This application claims the priority and benefit of U.S. Provisional Application No.

62/221190, filed September 21, 2015. The content of this application is incorporated herein by reference in its entirety.

Description of the text file submitted electronically

The contents of the electronically filed text file are incorporated herein in their entirety by reference: a copy of the sequence listing in machine-readable format (file name: APVO_052_01WO_SeqList_ST25.txt, date of entry: September 21, 2016, file size 544 kB).

Field of invention

The present invention relates to molecules that specifically bind to CD3, which may have at least one humanized CD3 binding domain. The CD3 binding protein therapeutic may be a protein therapeutic or a multispecific protein therapeutic. The multispecific protein therapeutic agent can bind subunits of both the tumor antigen and the CD3 T cell receptor complex on T cells to induce target-dependent cytotoxicity, T cell activation and proliferation.

State of the art invention

Targeting anti-CD3 antibodies to the T cell receptor (TCR) complex on human T cells has been proposed for the treatment of autoimmune disease and related disorders, for example for the treatment of organ allograft rejection. In addition to monospecific CD3-targeting therapeutics, multispecific polypeptides that bind selectively to both T cells and tumor cells may provide a mechanism for redirecting T cell cytotoxicity to tumor cells. Such multispecific polypeptides may be useful in the treatment of cancer. The clinical use of some anti-CD3 antibodies has been hampered by serious side effects. For example, OCTZ, a mouse monoclonal antibody specific for human CD3, induced T cell proliferation and cytokine production in vitro and resulted in massive cytokine release in vivo (Hirsch et al. (1989) J. Immunol 142: 737-43). The release of cytokines (also called cytokine storm) in turn led to a flu-like syndrome characterized by fever, chills, headaches, nausea, vomiting, diarrhea, respiratory distress, septic meningitis, and hypotension (Chatenoud (2003) Nature Reviews 3:123-132) .

There is a need for CD3 binding molecules that have improved thermal stability with a favorable profile in terms of manufacturing capability and reduced adverse events.

The essence of the invention

The present invention includes CD3 binding domains and polypeptides that have an advantageous manufacturing capability profile. Polypeptides described herein containing CD3 binding domains may be thermally stable. In some cases, the polypeptide has improved thermal stability compared to another CD3 binding polypeptide. The CD3 binding domains and polypeptides described herein may have reduced side effects (eg, may result in the release of low levels of cytokines when administered to a subject). In certain embodiments, the invention provides a CD3 binding domain that specifically binds to human CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is (a) at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical or at least about 99% identical to the amino acid sequence in SEQ ID NO: 88; or (b) at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 89; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 86. A CD3 binding domain may comprise an amino acid sequence that is at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 83 or SEQ ID NO: 84. CD3 binding domain may contain SEQ ID

NO: 83 or SEQ ID NO: 84.

In one embodiment, the CD3 binding domain comprises an immunoglobulin light chain variable region that comprises the LCDR1 amino acid sequence of SEQ ID NO: 94, the LCDR2 amino acid sequence of SEQ ID NO: 95, and the LCDR3 amino acid sequence of SEQ ID NO: 96, and the variable region an immunoglobulin heavy chain that comprises the amino acid sequence of HCDR1 according to SEQ ID NO: 91, the amino acid sequence of HCDR2 according to SEQ ID NO: 92, and the amino acid sequence of HCDR3 according to SEQ ID NO: 93. In another embodiment, the CD3 binding domain comprises an immunoglobulin light chain variable region , which contains the amino acid sequence of LCDR1 according to SEQ ID NO: 202, the amino acid sequence of LCDR2 according to SEQ ID NO: 203 and the amino acid sequence of LCDR3 according to SEQ ID NO: 204, and the variable region of the immunoglobulin heavy chain, which contains the amino acid sequence HCDR1 according to SEQ ID NO: 199, HCDR2 amino acid sequence according to SEQ ID NO: 200, and HCDR3 amino acid sequence according to SEQ ID NO: 201. In certain aspects, a CD3 binding domain may comprise an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, which contain framework regions, and at least one of an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region can be humanized. In one embodiment, the immunoglobulin light chain variable region comprises framework regions based on the human germline amino acid sequence IGKV3D-20*1. In another embodiment, the immunoglobulin heavy chain variable region comprises framework regions based on the amino acid sequence of the human germline IGHV1-69*02. In some embodiments, the amino acid residue at position 52 according to the CD3 binding domain immunoglobulin light chain variable region IMGT numbering system is arginine and/or the amino acid residue at position 53 according to the CD3 immunoglobulin light chain variable region IMGT numbering system. is tryptophan. The amino acid residue at position 27, according to the IMGT numbering system of the immunoglobulin heavy chain variable region of the CD3 binding domain, may be tyrosine. In some embodiments, the CD3 binding domain comprises one or more of the following: (a) the amino acid residue at position 9, according to the IMGT numbering system of the immunoglobulin heavy chain variable region, is a proline; (b) the amino acid residue at position 53 according to the IMGT numbering system of the immunoglobulin heavy chain variable region is isoleucine; and (c) the amino acid residue at position 21 according to the IMGT numbering system of the immunoglobulin light chain variable region is methionine. The amino acid residue at position 87 according to the IMGT numbering system of the immunoglobulin light chain variable region of the CD3 binding domain may be tyrosine. The amino acid residue at position 86 according to the IMGT numbering system of the immunoglobulin light chain variable region of the CD3 binding domain may be aspartic acid. In one embodiment, the amino acid residue at position 86 according to the IMGT numbering system of the CD3 binding domain immunoglobulin light chain variable region is aspartic acid, and the amino acid residue at position 87 according to the IMGT numbering system of the CD3 immunoglobulin heavy chain variable region is tyrosine. The invention also includes a CD3 binding domain which is a single chain variable fragment (scFv). In some aspects, the scFv may contain a linker between the heavy chain variable region and the light chain variable region. In one embodiment, the linker between the heavy chain variable region and the light chain variable region comprises the amino acid sequence QRHNNSSLNTGTQMAGHSPNS (SEQ ID NO: 148). In some embodiments, the scFv heavy chain variable region is amino-terminal to the scFv light chain variable region. In other embodiments, the scFv light chain variable region is amino-terminal to the scFv heavy chain variable region. The present invention includes a CD3 binding domain which has a thermal stability increased by at least 10% compared to that of a CD3 binding domain comprising a light chain variable region comprising SEQ ID NO: 90 and a heavy chain variable region comprising SEQ ID NO: 87. The median thermal transition point (Tm) of the CD3 binding domain may be increased by at least about 3°C, at least about 4°C, at least about 5°C, or at least about 6°C. and up to about 20° C. compared to the Tm of a CD3 binding domain comprising a light chain variable region comprising SEQ ID NO: 90 and a heavy chain variable region comprising SEQ ID NO: 87. The mid point thermal transition of a CD3 binding domain may be at least about 54°C, at least about 55°C, at least

- 2 042409 approximately 56°C or at least approximately 57°C and up to approximately 72°C. The thermal stability or midpoint of the thermal transition of the CD3 binding domain can be determined using differential scanning calorimetry or differential scanning fluorimetry. The CD3 binding domain described herein may have a storage stability increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least by about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and up to about 100% by compared with the storage stability of a CD3 binding domain containing a light chain variable region containing SEQ ID NO: 90 and a heavy chain variable region containing SEQ ID NO: 87. Storage stability can be determined after storage of the CD3 binding domain in PBS at approximately 25°C. In one embodiment, the CD3 binding domain is stable when stored in PBS at about 25° C. for at least about 6 days, at least about 10 days, or at least about 13 days, and up to about 90 days.

In some aspects, the CD3 binding domain described herein has a level of high molecular weight aggregates formed during recombinant expression that is at least about 5%, at least about 10%, at least about 20%, at least at least approximately 30% reduced and up to approximately 50% reduced compared to the level of high molecular weight aggregates formed during recombinant expression of the CD3 binding domain containing the light chain variable region containing SEQ ID NO: 90 and the heavy chain variable region containing SEQ ID NO: 87.

The invention also provides a CD3 binding domain that binds to human CD3 with an EC ₅₀ of about 10 nM or less. In some embodiments, the CD3 binding domain of the invention can also specifically bind to cynomolgus CD3. For example, a CD3 binding domain can bind to cynomolgus monkey CD3 with an EC ₅₀ of about 30 nM or less.

The present invention includes a CD3 binding domain that specifically binds to human CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, wherein (a) the immunoglobulin light chain variable region comprises an LCDR1 amino acid sequence according to SEQ ID NO: 94, an amino acid sequence LCDR2 according to SEQ ID NO: 95 and the amino acid sequence LCDR3 according to SEQ ID NO: 96, and the immunoglobulin heavy chain variable region contains the amino acid sequence HCDR1 according to SEQ ID NO: 91, the amino acid sequence HCDR2 according to SEQ ID NO: 92 and the amino acid sequence HCDR3 according to SEQ ID NO: 93; or (b) the immunoglobulin light chain variable region comprises the LCDR1 amino acid sequence of SEQ ID NO: 202, the LCDR2 amino acid sequence of SEQ ID NO: 203, and the LCDR3 amino acid sequence of SEQ ID NO: 204, and the immunoglobulin heavy chain variable region comprises the amino acid sequence of HCDR1 according to SEQ ID NO: 199, the amino acid sequence of HCDR2 according to SEQ ID NO: 200 and the amino acid sequence of HCDR3 according to SEQ ID NO: 201; and wherein the CD3 binding domain has one or more of the properties described herein. For example, (i) the midpoint thermal transition point of the CD3 binding domain (or protein containing the CD3 binding domain) is at least about 54°C, at least about 55°C, at least about 56°C, or at least about 57 °C and up to approximately 72°C; (ii) the CD3 binding domain (or protein containing the CD3 binding domain) is stable when stored in PBS at about 25° C. for at least about 6 days, at least about 10 days, or at least about 13 days, and up to approximately 90 days; (iii) a CD3 binding domain (or a protein containing a CD3 binding domain) binds to human CD3 with an EC ₅₀ of about 10 nM or less; and (iv) a CD3 binding domain (or a protein comprising a CD3 binding domain) binds to cynomolgus monkey CD3 with an EC ₅₀ of about 30 nM or less.

This invention also relates to a CD3-binding polypeptide containing any of the CD3-binding domains described herein. In some variations, the CD3 binding polypeptide may comprise an immunoglobulin constant region. This immunoglobulin constant region may contain immunoglobulin CH2 and CH3 domains from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain containing substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system. In certain embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain containing one or more substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system. In some embodiments, a CD3 binding polypeptide, when bound to a CD3 protein on a T cell, induces no or minimally detectable release of cytokines from said T cell. In certain aspects, the CD3 binding protein or polypeptide exhibits reduced cytokine release in a patient compared to cytokine release when an anti-CD3 OCTP antibody is administered to the patient. In some instances, a CD3 binding polypeptide can induce T cell activation or T cell proliferation.

In certain aspects, the CD3 binding polypeptide further comprises a second binding domain. The second binding domain may be a single chain variable fragment (scFv). In some embodiments, the second binding domain binds or interacts with a tumor antigen (eg, PSMA, CD19, CD20, CD37, CD38, CD123, Her2, ROR1, RON, A33 glycoprotein antigen (gpA33), or CEA).

The invention further includes a CD3 binding polypeptide comprising: (i) a CD3 binding domain and (ii) a second binding domain. In some embodiments, the CD3 binding polypeptide comprises, in amino-terminal to carboxy-terminal order or in carboxy-terminal to amino-terminal order, (i) a CD3 binding domain, (ii) a hinge region, and (iii) an immunoglobulin constant region. In some embodiments, a CD3 binding polypeptide comprises, in amino-terminal to carboxy-terminal order, (i) a second binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxy-terminal linker, and (v) CD3 binding domain. In other embodiments, the CD3 binding polypeptide comprises, in order from carboxy-terminus to amino-terminus, (i) a second binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an amino-terminal linker, and ( v) CD3 binding domain. In certain variations, the first and/or second binding domain is scFv. Non-limiting examples of carboxy-terminal and amino-terminal linkers include flexible linkers containing glycine-serine (eg, (Gly4Ser)) repeats and linkers derived from (i) a type II C lectin stem region or (ii) an immunoglobulin hinge region. In certain aspects, the carboxy-terminal linker (or amino-terminal linker) comprises or consists of SEQ ID NO: 196. In certain aspects, the invention provides a CD3 binding polypeptide (e.g., multispecific) wherein (i) the CD3 binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1, LCDR2 and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; and (ii) the second binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1, LCDR2 and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2 and HCDR3.

The present invention includes a CD3 binding polypeptide that induces redirected T cell cytotoxicity (PTCC). For example, a CD3 binding polypeptide can induce PTCC with an EC ₅₀ of about 30 pM or less. In some embodiments, the CD3 binding polypeptide exhibits no or minimal antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CCC) activity. In certain aspects, null ADCC and/or CDC activity is obtained through mutations in the hinge region and constant region of an Ig (eg, Fc).

The CD3 binding polypeptide may further comprise an immunoglobulin heterodimerization domain. In some embodiments, an immunoglobulin heterodimerization domain comprises an immunoglobulin CH1 domain or an immunoglobulin CL domain. In some aspects, the CD3 binding polypeptide is a heterodimeric CD3 binding protein comprising (i) a first polypeptide chain comprising, in amino-terminal to carboxy-terminal or carboxy-terminal to amino-terminal order, (a) a CD3 binding domain , which specifically binds human CD3, (b) a first hinge region, (c) a first immunoglobulin constant region, and (d) a first immunoglobulin heterodimerization domain; and (ii) a second polypeptide chain comprising, in order from amino-terminus to carboxy-terminus or from carboxy-terminus to amino-terminus, (a') a second hinge region, (b') a second immunoglobulin constant region, and (c' ) a second immunoglobulin heterodimerization domain that differs from the first immunoglobulin heterodimerization domain of the first single chain polypeptide, wherein the first and second immunoglobulin heterodimerization domains bind to each other to form a heterodimer. In one embodiment, the first immunoglobulin heterodimerization domain comprises an immunoglobulin CH1 domain and the second immunoglobulin heterodimerization domain comprises an immunoglobulin CL domain, or the first immunoglobulin heterodimerization domain comprises an immunoglobulin CL domain and the second immunoglobulin heterodimerization domain comprises an immunoglobulin CH1 domain. At least one of the first and second immunoglobulin constant regions may comprise the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or any combination thereof; an immunoglobulin CH3 domain of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM, or any combination thereof; or immunoglobulin CH3 and CH4 domains of IgE, IgM, or combinations thereof. In some aspects, the second polypeptide chain of the heterodimeric CD3 binding protein may further comprise a second binding domain. In certain embodiments, the second binding domain may be amino-terminal or carboxy-terminal to the second hinge region.

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In some variations, the CD3 binding polypeptide may be a bispecific single chain antibody molecule comprising a CD3 binding domain and a second binding domain, the binding domains being in the order VH CD3-VL CD3-second binding domain VH-second binding domain VL, or VL CD3 -VH CD3-second binding domain VH second binding domain VL, or second binding domain VH-second binding domain VL-VH CD3-VL CD3, or second binding domain VH-second binding domain VL-VL CD3-VH CD3. The invention also provides an isolated nucleic acid molecule encoding a CD3 binding domain or a CD3 binding polypeptide as described herein, or a portion of said CD3 binding domain or polypeptide. In some aspects, the invention includes an expression vector comprising a nucleic acid segment encoding the CD3 binding domain or CD3 binding polypeptide described herein, wherein the nucleic acid segment is operably linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell. Included in the invention is a recombinant host cell containing an expression vector.

The present invention includes an expression vector comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric CD3-binding polypeptide, and wherein the first and second nucleic acid segments are functionally associated with regulatory sequences suitable for the expression of nucleic acid segments in the host cell. Part of this invention is a recombinant host cell containing an expression vector containing first and second expression units.

The present invention further relates to a method for producing a CD3 binding polypeptide, comprising: culturing a recombinant host cell containing an expression vector as described herein under conditions in which expression of a nucleic acid segment occurs and thereby obtaining a CD3 binding polypeptide. In some embodiments, a method for producing a heterodimeric CD3-binding protein comprises: culturing a recombinant host cell comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of the heterodimeric CD3- binding protein, wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for the expression of the nucleic acid segments in the host cell, and said cultivation occurs under conditions in which the expression of the first and second nucleic acid segments occurs, and the encoded the polypeptide chains are obtained as a heterodimeric CD3 binding protein. These methods may further include isolating the CD3 binding polypeptide or heterodimeric CD3 binding protein.

The present invention includes a pharmaceutical composition comprising a CD3 binding polypeptide as described herein and a pharmaceutically acceptable carrier, diluent or excipient. The present invention also relates to a method for inducing redirected T-cell cytotoxicity (PTCC) against cells expressing a tumor-associated antigen, comprising: bringing said tumor-associated antigen-expressing cell into contact with a CD3-binding polypeptide, said contacting occurring under conditions in which PTCC against tumor-associated antigen-expressing cells. One aspect of the invention includes a method of inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD3 binding polypeptide or pharmaceutical composition described herein. This invention includes a method of treating cancer or an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD3-binding polypeptide or a pharmaceutical composition described herein. Non-limiting examples of cancers that can be treated with the methods and CD3 binding polypeptides described herein include prostate cancer, colorectal cancer, renal cell carcinoma, bladder cancer, salivary gland cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, breast cancer (eg, triple-negative breast cancer), melanoma, adrenal cancer, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute myelocytic leukemia (AML), B-lymphoid leukemia, plasmacytoid dendritic cell blast (BNPDC) and hairy cell leukemia.

This invention includes a CD3 binding domain that specifically binds to human CD3 and contains SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60. The invention also relates to a CD3 binding protein which is a dimer of two identical polypeptides, each polypeptide is any of the CD3 binding polypeptides described herein.

These and other embodiments and/or other aspects of the invention will become apparent upon examination of the following detailed description of the invention and the accompanying drawings.

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Brief description of graphic materials

Fig. 1 (upper panel) is a graph showing the results of flow cytometry determination of the binding of CD3 binding domain constructs to Jurkat T cells. The y-axis shows the average fluorescence intensity (MIF) of bound molecules on live cells. Concentration (nM) of CD3 binding domain constructs is shown on the x-axis. The table (bottom panel) shows the EC ₅₀ values obtained from the graph.

Fig. 2 (upper panel) is a graph showing the results of flow cytometric determination of the binding of CD3 binding domain constructs to a subclone of the Jurkat T cell line with higher levels of CD3 expression. The y-axis shows the average fluorescence intensity (MIF) of bound molecules on living cells. Concentration (nM) of CD3 binding domain constructs is shown on the x-axis. The table (bottom panel) shows the EC ₅₀ values obtained from the graph.

Fig. 3 is a graph showing the results of differential scanning fluorimetry studies conducted on CD3 binding domain constructs. The figure also shows the values of the midpoint of the thermal transition (Tm), obtained from the graph.

Fig. 4 is a graph showing the results of flow cytometric determination of the binding of CD3 binding domain constructs to a subclone of the Jurkat T cell line with higher levels of CD3 expression. The y-axis shows the average fluorescence intensity (MIF) of bound molecules on live cells. Concentration (nM) of CD3 binding domain constructs is shown on the x-axis.

Fig. 5 is a graph showing the results of flow cytometric determination of the binding of CD3 binding domain constructs to a subclone of the Jurkat T cell line with higher levels of CD3 expression. The y-axis shows the average fluorescence intensity (MIF) of bound molecules on living cells. Concentration (nM) of CD3 binding domain constructs is shown on the x-axis.

Fig. 6 (upper panel) is a graph showing the results of the chromium-51 release assay testing the efficacy of bispecific anti-PSMA and anti-CD3 constructs in inducing target-dependent T-cell cytotoxicity with human PBMCs at 4 hours against C4-2B cells. The y-axis shows the percentage of specific lysis compared to the control total lysis. Concentration (pM) of CD3 binding domain constructs is shown on the x-axis. The table (bottom panel) shows the EC ₅₀ values obtained from the graph.

Fig. 7A and 7B are graphs showing the results of flow cytometry determination of the binding of CD3 binding domain constructs to cynomolgus monkey T cells. Each graph shows the y-axis mean fluorescence intensity (MFI) of bound molecules on live cells. The concentration (nM) of the CD3 binding domain constructs is shown on each graph along the x-axis.

Fig. 8A and 8B are graphs showing the results of the chromium-51 release assay determining the efficacy of CD3 binding domain constructs in inducing cynomolgus monkey PBMC target-dependent T-cell cytotoxicity at 4 hours against C4-2B cells. In each graph, the y-axis shows the percentage of specific lysis compared to the control total lysis. The concentration (pM) of the CD3 binding domain constructs is shown on each graph along the x-axis.

Fig. 9 is a graph showing the results of flow cytometric determination of the binding of bispecific anti-CD37 and anti-CD3 constructs to cynomolgus monkey T cells. The y-axis shows the average fluorescence intensity (MIF) of bound molecules on live cells. Concentration (nM) of CD3 binding domain constructs is shown on the x-axis. TSC394 DY refers to the TSC394 design including the E86D and F87Y replacements.

Fig. 10 (upper panel) is a graph showing the results of the chromium-51 release assay testing the efficacy of bispecific anti-CD37 and anti-CD3 constructs in inducing target-dependent T-cell cytotoxicity in cynomolgus monkey PBMC at 4 hours against Ramos cells. In each graph, the y-axis shows the percentage of specific lysis compared to the control total lysis. The concentration (pM) of the CD3 binding domain constructs is shown on each graph along the x-axis. The table (bottom panel) shows the EC ₅₀ values obtained from the graph. TSC394 DY refers to the TSC394 design including the E86D and F87Y replacements.

Fig. 11 is a graph showing the results of determining the storage stability of CD3 binding domain constructs at 25°C for the number of days indicated on the x-axis. CAS105 is a control construct containing the CD3 binding domain of DRA222.

Fig. 12 is a graph showing serum stability results in human serum for anti-ROR1 x anti-CD3 bispecific molecules over the number of days indicated on the x-axis.

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In FIG. 13 shows the amino acid sequence alignment of DRA222 scFv (SEQ ID

NO: 85), TSC455 scFv (SEQ ID NO: 83) and TSC456 scFv (SEQ ID NO: 84). The sequences of these structures are also given in table. 14.

Fig. 14 is a graph showing the results of determining the serum concentration of anti-PSMA X anti-CD3 bispecific binding molecules in BALB/c mice as a function of time. Results are expressed as mean serum concentration (µg/mL) over time for each of the treatment groups. Each point shows the mean (+ standard deviation) for individual animals.

Fig. 15 is a table showing a comparison of the pharmacokinetic parameters of anti-PSMA X anti-CD3 bispecific binding molecules in BALB/c mice.

Fig. 16 is a graph showing the results of determining the serum concentration of anti-ROR1 X anti-CD3 bispecific binding molecules in NSG mice as a function of time. Results are expressed as mean serum concentration (μg/mL) over time for each of the treatment groups.

Fig. 17A and 17B are tables showing a comparison of the pharmacokinetic parameters of anti-ROR1 X anti-CD3 bispecific binding molecules in NSG mice. In FIG. 17A shows estimated pharmacokinetic parameters calculated using NCA for IV dosing, with random sampling and constant weighting for each treatment group. In FIG. 17B shows the estimated pharmacokinetic parameters determined using the 2-compartment V/V model with 1/Y weighting for each treatment group.

Fig. 18 is a graph showing the results of an analysis of the effect of anti-ROR1 X antu-CD3 bispecific binding molecules on the growth of MDA-MB-231 xenograft tumor.

Fig. 19 is a graph showing the results of an analysis of the effect of anti-ROR1 X antu-CD3 bispecific binding molecules on Kasumi-2 xenograft tumor growth. Results are shown for human T cell donor #336.

Detailed description of the invention

The present invention provides binding domains that specifically bind to CD3 (differentiation cluster 3) and binding molecules (eg polypeptides and proteins) that specifically bind to CD3. These binding molecules can specifically bind to CD3 and at least one other target. In some embodiments, the CD3 binding molecule described herein has a favorable production capability profile characterized by one or more of the properties described below. In certain embodiments, CDRs from the anti-CD3 antibody Cris7 have been used to construct CD3 binding molecules with improved and novel properties. Accordingly, the present invention provides humanized anti-CD3 binding domains and proteins having improved properties (e.g., thermal stability, storage stability, serum half-life, reduced high molecular weight aggregate formation) compared to other anti-CD3 binding domains and proteins. In some aspects of the invention, the CD3 binding molecule is thermostable. For example, said molecule may have improved thermal stability compared to another CD3 binding molecule (eg, DRA222). CD3 binding molecules can be characterized by high production yield and long serum half-life and long storage half-life. In addition, the CD3 binding molecules described herein may have a low risk of adverse side effects when administered to a subject. For example, CD3 binding molecules can lead to the release of low levels of cytokines.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the specified range and, where appropriate, its fractional values (such as one tenth and one hundredth of an integer), if not otherwise specified. It should be understood that in the context of this document, the singular refers to one or more of the numbered components, unless otherwise indicated. The use of an alternative (eg, or) should be understood to mean one, both, or any combination of the alternatives. In the context of this document, the terms include and contain are used interchangeably. In addition, it should be understood that polypeptides containing various combinations of components (eg, domains or regions) and substituents described herein are disclosed in this application to the same extent as if each polypeptide were listed individually. Thus, the selection of specific components of individual polypeptides is within the scope of the present invention.

All documents or portions of documents cited in this text, including but not limited to patents, patent applications, articles, books and treatises, are expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the included documents or portions of documents provide a definition of a term that conflicts with the definition of that term in this application, the definition given in this application takes precedence. At the same time, the mention of any reference material, article, publication, patent, patent publication and application for

- 7 042409 patent cited in this document is not and should not be taken as an admission, or any form of suggestion that they constitute the actual state of the art or form part of the well-known state of the art in any country in the world.

As used herein, a binding domain or binding region refers to a domain, region, portion, or region of a protein, polypeptide, oligopeptide, or peptide, or an antibody, or a binding domain derived from an antibody, that has the ability to specifically recognize and bind a target molecule, such as antigen, ligand, receptor, substrate, or inhibitor (eg, CD3). Examples of binding domains include single chain antibody variable regions (eg, domain antibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (eg, cytokines, chemokines). In certain embodiments, the binding domain comprises or consists of an antigen-binding region (e.g., comprising a heavy chain variable sequence and a light chain variable sequence, or three light chain complementarity determining regions (CDRs) and three heavy chain CDRs from an antibody placed in alternative framework regions (FRs). ) (e.g., human FRs, optionally containing one or more amino acid substitutions.) Many assays are known to determine the binding domains of the present invention that specifically bind to a particular target, including Western blotting, ELISA, phage display library screening, and BIACORE® interaction analysis. In the context of this document, a CD3 binding polypeptide may have a first binding domain and optionally a second binding domain.In certain embodiments, the first binding domain is a CD3 binding domain in antibody or antibody-like format. lka or domain. In certain embodiments that include both the first and second binding domains, the second binding domain is a tumor antigen binding domain. In other embodiments, the second binding domain is a second CD3 binding domain. In other embodiments, the second binding domain is a binding domain other than a tumor antigen binding domain.

A binding domain or protein specifically binds a target if it binds the target with affinity or K _acc . (i.e., the equilibrium association constant of a particular binding interaction, measured in units of 1/M) equal to or greater than 105 M-1, while not showing significant binding of other components present in the test sample. Binding domains can be classified as high affinity binding domains and low affinity binding domains. High affinity binding domains include binding domains with K _acc . of at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 or at least 10 ¹³ M ^-1 . Low affinity binding domains include binding domains with Kacc., comprising up to 10 ⁷ M ^-1 , up to 10 ⁶ M ^-1 , up to 105 M ^-1 . Alternatively, affinity can be determined using the equilibrium dissociation constant (KdCC.) of a particular binding interaction, measured in units of M (eg, from 10 ^-5 M to 10 ^-13 M). The affinity of the binding domain-containing polypeptides and single-chain polypeptides of the present invention can be readily determined using conventional methods (see, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and US Pat. Nos. 5,283,173, 5468614 or equivalent references). In the art, CD3 is known as a six-chain multiprotein complex (see, for example, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999) that are subunits of the T cell receptor complex. In mammals, the CD3 subunits of the T cell receptor complex are a CD3y chain, a CD35 chain, two CD3ε chains, and a homodimer of CD3Z chains. The CD3y, CD3δ and CD3ε chains are highly related cell surface proteins from the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD3δ, and CD3ε chains are negatively charged, allowing these chains to bind to positively charged T-cell receptor chains. The intracellular tails of each of the CD3y, CD3δ, and CD3ε chains contain one conserved motif known as the immunoreceptor tyrosine activating motif, or ITAM, while each CD3Z chain contains three such motifs. ITAMs are believed to be important for the ability of the TKR complex to transmit signals. The CD3s used in the present invention may be from various animal species, including humans, monkeys, mice, rats, or other mammals. In the context of this document, a conservative substitution in the art is the replacement of one amino acid with another amino acid that has similar properties. Examples of conservative substitutions are well known in the art (see, for example, WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp .71-77 Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8. In certain embodiments, a conservative substitution includes substitution of a leucine for a serine.

In the context of this document, the term derivative refers to the modification of one or more amino acid residues of a peptide by chemical or biological means, with or without the use of an enzyme, for example, by glycosylation, alkylation, acylation, formation

- 8 042409 ester or amide formation.

In the context of this document, the polypeptide or amino acid sequence derived from the specified polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, a polypeptide or amino acid sequence derived from a particular sequence (sometimes referred to as a start or parent sequence) has an amino acid sequence that is substantially identical to the start sequence or portion thereof, said portion consisting of at least 10-20 amino acids, at least at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identified by one of skill in the art as being derived from a start sequence. For example, the binding domain may be derived from an antibody, such as Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc. Polypeptides derived from another polypeptide may contain one or more mutations in relative to the starting polypeptide, for example, one or more amino acid residues substituted with another amino acid residue, or contain one or more insertions or deletions of amino acid residues. The polypeptide may contain an amino acid sequence that is not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity to the starting polypeptide. In one embodiment, the variant has an amino acid sequence having from about 60% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide. In another embodiment, the variant has an amino acid sequence having from about 75% to less than 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.

In the context of this document, unless otherwise indicated, the position of an amino acid residue in the variable region of an immunoglobulin molecule is numbered according to the IMGT criterion (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508), amino acid residue position in the constant region, the immunoglobulin molecule is numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). The Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest, ^5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)) is an alternative system used to designate the position of an amino acid residue in the variable region of an immunoglobulin molecule. As used herein, the term dimer refers to a biological compound that is composed of two subunits bonded to each other via one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonding and hydrophobic interactions) and is stable under appropriate conditions (eg, under physiological conditions, in an aqueous solution suitable for expression, purification and/or storage of recombinant proteins, or under conditions for non-denaturing and/or non-reducing electrophoresis). As used herein, a heterodimer or heterodimeric protein refers to a dimer formed by two different polypeptides. The heterodimer does not include an antibody formed by four polypeptides (ie two light chains and two heavy chains). As used herein, a homodimer or homodimeric protein refers to a dimer formed by two identical polypeptides.

In some embodiments, the CD3 binding polypeptide comprises, in amino-terminal to carboxy-terminal order or in carboxy-terminal to amino-terminal order, (i) a second binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxy-terminal linker (or amino-terminal linker); and (v) a CD3 binding domain. As used herein, and depending on the context, a hinge region or hinge refers to a polypeptide region between a binding domain (eg, a CD3 binding domain or a second binding domain) and an immunoglobulin constant region. As used herein and depending on the context, a linker may refer to (1) a polypeptide region between the V _H and V _L regions in a single chain Fv (scFv) or (2) a polypeptide region between an immunoglobulin constant region and a second binding domain in a CD3 binding polypeptide, containing two binding domains. The polypeptide region between the immunoglobulin constant region and the CD3 binding domain in a CD3 binding polypeptide containing two binding domains may also be referred to as a carboxy-terminal linker or an amino-terminal linker. Non-limiting examples of carboxy-terminal and amino-terminal linkers include flexible linkers containing glycine-serine repeats and linkers derived from (a) the interdomain region of a transmembrane protein (eg, a type I transmembrane protein); (b) a type II C lectin stem region; or (c) the hinge region of an immunoglobulin. Non-limiting examples of hinge regions and linkers are given in table. 1 and 2. In some embodiments, the linker provides a spacer function compatible with the interaction of the two binding subdomains such that the resulting polypeptide retains the affin

- 9 042409 specific binding to the same target molecule as an antibody that contains the same light and heavy chain variable regions. In certain embodiments, the linker is five to about 35 amino acids, such as about 15 to about 25 amino acids. Wild-type immunoglobulin hinge refers to naturally occurring upper and lower hinge amino acid sequences located between and connecting the CH1 and CH2 domains (in the case of IgG, IgA and IgD) or located between and connecting the CH1 and CH3 domains (in the case of IgE and IgM) located in the heavy chain of the antibody. In certain embodiments, the wild-type immunoglobulin hinge sequence is human and may comprise a human IgG hinge. An altered wild-type immunoglobulin hinge or an altered immunoglobulin hinge refers to (a) a wild-type immunoglobulin hinge having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions). ), or (b) a portion of a wild-type immunoglobulin hinge region having a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, or 20 amino acids) to about 120 amino acids (e.g., having a length of about 10 to about 40 amino acids, or about 15 to about 30 amino acids, or about 15 to about 20 amino acids, or about 20 to about 25 amino acids), having up to about 30% amino acid changes (for example, up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions, or a combination thereof) and containing a major hinge region IgG described in US 2013/0129723 and US 2013/0095097.

As used herein, the term humanized refers to a process designed to make an antibody or immunoglobulin binding proteins and polypeptides derived from a non-human species (e.g. mouse or rat) less immunogenic in humans while retaining the antigen-binding properties of the original antibody, using genetic engineering methods. In some embodiments, the binding domain(s) of the antibody or immunoglobulin binding proteins and polypeptides (eg, light and heavy chain variable regions, Fab, scFv) are humanized. Non-human binding domains can be humanized using methods known as CDR grafting (Jones et al., Nature 321:522 (1986)), and variants thereof, including remodeling (Verhoeyen et al., 1988 Science 239:1534-1536; Riechmann et al., 1988 Nature 332:323-337; Tempest et al., Bio/Technol. 1991 9:266-271), hyperchimerization (Queen et al., 1989 Proc. Natl. Acad. Sci. USA 86:10029-10033 ; Co et al., 1991 Proc. Natl. Acad. Sci. USA 88:2869-2873; Co et al., 1992 J. Immunol. 148:1149-1154) and veneering (Mark et al., Derivation of therapeutically active humanized and veneered anti-CD18 antibodies. In: Metcalf B. W., Dalton B. J., eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). When derived from a non-human source, other regions of the antibody or immunoglobulin binding proteins and polypeptides, such as the hinge region and constant region domains, may also be humanized.

As used herein, an immunoglobulin dimerization domain or an immunoglobulin heterodimerization domain refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts with or binds to a different immunoglobulin domain of a second polypeptide chain, where the interaction of different immunoglobulin heterodimerization domains substantially contributes to or effectively promotes heterodimerization of the first and second polypeptide chains ( i.e. the formation of a dimer between two different polypeptide chains, which is also called a heterodimer). The interaction between the immunoglobulin heterodimerization domains significantly contributes to or effectively stimulates the heterodimerization of the first and second polypeptide chains if there is a statistically significant decrease in dimerization between the first and second polypeptide chains in the absence of an immunoglobulin heterodimerization domain of the first polypeptide chain and/or an immunoglobulin heterodimerization domain of the second polypeptide chain. In certain embodiments, if the first and second polypeptide chains are expressed together, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least about 80 % to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the first and second polypeptide chains form heterodimers with each other. Exemplary immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g., Ck and Cλ isotypes), or derivatives thereof, including CH1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains as provided herein. . An immunoglobulin constant region or constant region is a term which, as defined herein, refers to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant region domains. In certain embodiments, the immunoglobulin constant region corresponds to or is derived from part or all of one or more constant region domains, but not all of the source antibody's constant region domains. In certain embodiments, the constant region contains IgG CH2 and CH3 domains,

- 10 042409 for example the CH2 and CH3 domains of IgG1. In certain embodiments, the constant region does not contain a CH1 domain. In certain embodiments, the constant region domains that make up the constant region are human. In some embodiments (e.g., certain variations of the CD3 binding protein or polypeptide), the constant region domains of the fusion protein of this invention have no or minimal effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation and complement-dependent cytotoxicity (CCC), while maintaining this ability to bind certain Fc receptors (such as FcRn, neonatal Fc receptor) and maintain a relatively long in vivo half-life. In other variations, the fusion protein of this invention contains constant domains that retain such effector function of one or both of ADCC and CDC. In certain embodiments, the binding domain of this invention is fused to a human IgG1 constant region, wherein the IgG1 constant region contains one or more of the following mutated amino acids: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 ( G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (EU numbering). For example, one or more of these amino acids may be substituted for alanine. In a further embodiment, the IgG1 Fc domain contains each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to alanine (i.e., L234A, L235A, G237A, E318A, K32OA, and K322A, respectively ), and also, optionally, an N297A mutation (i.e., virtually eliminating glycosylation of the CH2 domain).

An Fc region or Fc domain refers to a polypeptide sequence corresponding to or derived from the portion of the parent antibody that is responsible for binding to antibody receptors on cells and the C1q complement component. Fc means a crystallizable fragment, an antibody fragment that readily forms a protein crystal. Different protein fragments, which were originally described using proteolytic cleavage, can determine the overall structure of the immunoglobulin protein. According to the original definition in the literature, the Fc fragment consists of disulfide-linked heavy chain hinge regions, CH2 and CH3 domains. However, the term has recently been applied to a single chain consisting of CH3, CH2 and at least part of the hinge region, which is sufficient to form a disulfide-linked dimer with a second such chain. For a review of the structure and function of immunoglobulin, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes naturally occurring sequence variants.

In some embodiments, the CD3 binding protein comprises a protein backbone as generally described in, for example, US Patent Application Publication Nos. 2003/0133939, 2003/0118592, and 2005/0136049. A CD3 binding protein may comprise, in amino-terminal to carboxy-terminal order, a first binding domain, a hinge region, and an immunoglobulin constant region. In other embodiments, the CD3 binding protein comprises a protein backbone as generally described, for example, in US Patent Application Publication No. 2009/0148447. A CD3 binding protein may comprise, in order from carboxy-terminus to amino-terminus, an immunoglobulin constant region, a hinge region, and a first binding domain. The CD3 binding polypeptides and proteins described herein may include a multispecific binding protein backbone. Multispecific binding proteins and polypeptides that use scaffolds are described, for example, in PCT Application Publication No. WO 2007/146968, US Patent Application Publication No. 2006/0051844, PCT Application Publication No. WO 2010/040105, PCT Application Publication No. WO 2010/040105, PCT Application Publication No. No. WO 2010/003108, US patent No. 7166707 and US patent No. 8409577, which are all incorporated herein by reference in their entirety. A CD3 binding protein may contain two binding domains (the domains may be designed to specifically bind to the same or different targets), two hinge regions, and an immunoglobulin constant region. The CD3 binding protein may be a homodimeric protein containing two identical disulfide-linked polypeptides. As used herein, a type II C lectin stem region refers to the portion of the extracellular domain of a type II C lectin located between the C-type lectin-like domain (CTLD; eg, natural killer cell receptor-like CTLD) and the transmembrane domain. For example, in the human CD94 molecule (GenBank Accession No. AAC50291.1, PRI November 30, 1995), the extracellular domain corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. Accordingly, the human CD94 stem region contains amino acid residues 34-60 that lie between the membrane and CTLD (see Boyington et al., Immunity 10:75, 1999; for other stem regions, see also Beavil et al., Proc Nat'l Acad Sci USA 89:753, 1992; and Figdor et al., Nature Rev. Immunol. 2:77, 2002). These type II C lectins can also contain six to 10 junction amino acids between the stem region and the transmembrane region of the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1, PRI June 15, 2010) contains a transmembrane domain within amino acids 71-93 and an extracellular domain within amino acids 94-233. CTLD consists of amino acids 119-231 and the stem region contains amino acids 99-116 which are flanked by compounds

- 11 042409 of five and two amino acids. Other type II C lectins, as well as their extracellular ligand-binding domains, interdomain or stem regions, and CTLDs, are known in the art (see, for example, GenBank Accession No. NP_001993.2; AAH07037.1, PRI July 15, 2006 ; NP_001773.1, PRI Jun 20, 1010; AAL65234.1, PRI Jan 17, 2002, and CAA04925.1, PRI Nov 14, 2006, for human CD23, CD69, CD72, NKG2A, and NKG2D sequences and descriptions, respectively) . As used herein, the interdomain region of a transmembrane protein (eg, type I transmembrane protein) refers to the portion of the extracellular domain of a transmembrane protein located between two adjacent domains. Examples of interdomain regions include regions that bind adjacent Ig domains of members of the immunoglobulin superfamily (e.g., an immunoglobulin hinge region of IgG, IgA, IgD, or IgE; a region that binds IgV and IgC2 domains in CD2; or a region that binds IgV and IgC domains in CD80 or CD86 ). Another example of an interdomain region is the region binding the non-Ig domain and the IgC2 domain of CD22, which binds the sialic acid type I Ig-like lectin.

A polypeptide region derived from a type II C lectin stem region or derived from an interdomain region of a transmembrane protein (e.g., an immunoglobulin hinge region) refers to from about five to about 150 amino acid sequence, from about 5 to about 100 amino acid sequence, from about 5 to about 50 amino acid sequence, from about 5 to about 40 amino acid sequence, from about 5 to about 30 amino acid sequence, from about 5 to about 25 amino acid sequence, from about 5 to about 20 amino acid sequence, from about 10 to about 25 amino acid sequence, from about 10 to about 20 amino acid sequence, from about 8 to about 20 amino acid sequence, from about 9 to about 20-a about 10 to about 20 amino acid sequence, about 11 to about 20 amino acid sequence, about 12 to about 20 amino acid sequence, about 13 to about 20 amino acid sequence, about 14 to about 20 amino acid sequence , from about 15 to about 20 amino acid sequences, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid sequences, and all or at least part of it contains (i) a wild-type sequence of the stem region or interdomain region; (ii) a fragment of the sequence of the wild-type stem region or interdomain region; (iii) a polypeptide having at least 80%, 85%, 90% or 95% amino acid sequence identity with any of (i) or (ii); or (iv) any of (i) or (ii) in which one, two, three, four, or five amino acids have a deletion, insertion, or substitution, or any combination thereof, e.g., one or more changes are substitutions, or one or more mutations include only one deletion. In some embodiments, a stem region derivative is more resistant to proteolytic cleavage than a wild-type stem region sequence, such as those derived from about eight to about 20 amino acids NKG2A, NKG2D, CD23, CD64, CD72, or CD94.

As used herein, junction amino acids or junction amino acid residues refer to one or more (e.g., about 2-10) amino acid residues between two adjacent regions or domains of a polypeptide, such as between a hinge and an adjacent immunoglobulin constant region, or between a hinge and an adjacent binding domain. , or between a peptide linker and an adjacent immunoglobulin variable domain or adjacent immunoglobulin constant region. Connecting amino acids may result from the construction of the polypeptide (eg, amino acid residues resulting from the use of a restriction enzyme site during construction of the nucleic acid molecule encoding the polypeptide). As used herein, a linker between CH3 and CH1 or CL refers to one or more (e.g., about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8-10, about 9-10, about 8-12, about 9-12, or about 10-12) amino acid residues between the C-terminus of the CH3 domain (e.g. wild-type or mutated CH3) and the N-terminus of the CH1 domain, or CL domain (for example, Sk).

As used herein, the term patient or subject in need refers to a patient or subject at risk for or suffering from a disease, disorder or condition that is treatable or ameliorated by a CD3 binding protein or polypeptide or composition provided herein. . As used herein, the term pharmaceutically acceptable refers to molecular compounds and compositions that generally do not cause allergic or other serious adverse reactions when administered by methods well known in the art. Molecular compounds and compositions approved by a federal or state government regulatory agency or listed in the USP or other generally accepted pharmacopoeias for use in animals, and in particular in humans, are considered pharmaceutically acceptable.

In the context of this document, the term promoter refers to the region of DNA involved in the binding of RNA polymerase to initiate transcription. In the context of this document, the terms nucleic acid, nucleic acid molecule or polynucleotide refers to deoxyribonucleotides or ribonucleotides and their polymers in single or double stranded form. Unless specifically limited, these terms include nucleic acids containing analogs of naturally occurring nucleotides that have the same binding properties as a standard nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleotide sequence, of course, also includes conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. In particular, degenerate codon substitutions can be made by creating sequences in which the third position of one or more selected (or all) codons is replaced by residues with mixed bases and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081 Ohtsuka et al (1985) J Biol Chem 260:2605-2608 Cassol et al (1992) Rossolini et al (1994) Mol Cell Probes 8:91-98). The term nucleic acid is used interchangeably with a gene, cDNA, and mRNA encoded by a gene. As used herein, the terms nucleic acid, nucleic acid molecule, or polynucleotide are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA made using nucleotide analogs, as well as their derivatives, fragments and homologues.

The term expression refers to the biosynthesis of the product encoded by the nucleic acid. For example, in the case of a nucleic acid segment encoding a polypeptide of interest, expression includes transcription of the nucleic acid segment into mRNA and translation of the mRNA into one or more polypeptides. The terms expression unit and expression cassette are used interchangeably herein and refer to a nucleic acid segment encoding a polypeptide of interest and capable of allowing expression of the nucleic acid segment in a host cell. An expression unit typically contains a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator that are in a functional configuration. In addition to the transcription promoter and terminator, the expression unit may additionally contain other nucleic acid segments, such as, for example, an enhancer or a polyadenylation signal. In the context of this document, the term expression vector refers to a nucleic acid molecule, linear or circular, containing one or more expression units. In addition to one or more expression units, the expression vector may also contain additional nucleic acid segments, such as, for example, one or more origins of replication or one or more selectable markers. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

In the context of this document, the term sequence identity refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. If a position in one sequence is occupied by the same nucleotide base or amino acid residue as the corresponding position in the compared sequence, the sequences are said to be identical at that position. Percent sequence identity is calculated by determining the number of positions in which both sequences contain an identical nucleotide base or amino acid residue to obtain the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percent sequence identity. The percent sequence identity is determined by comparing two sequences optimally aligned in the comparison window. The comparison window for nucleotide sequences may be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids. The comparison window for polypeptide sequences may be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids. In order to optimally align sequences for comparison, a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions, called gaps, while the standard sequence remains unchanged. The optimal alignment is the alignment that, even in the presence of gaps, gives the largest possible number of identical positions between the standard and the compared sequence. The percent sequence identity between two sequences can be determined using the version of the BLAST 2 Sequences software available from the National Center for Biotechnology Information on September 1, 2004, which includes the BLASTN (for comparison of nucleotide sequences) and BLASTP (for comparison of polypeptide sequences) programs.

- 13 042409 sequences) which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When applying BLAST 2 Sequences, you can use the parameters that were the default parameters on September 1, 2004, with word length (3), gap opening penalty (11), gap extension penalty (1), gap closing penalty (50 ), the expected value (10), and any other required parameter, including but not limited to the matrix option. Two nucleotide or amino acid sequences are considered to have substantially the same sequence identity or substantial sequence identity if the two sequences share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to each other. As used herein, a polypeptide or polypeptide chain is a uniform, linear and continuous arrangement of covalently linked amino acids. It does not include two polypeptide chains linked to each other in a non-linear manner, eg via an interchain disulfide bond (eg, half an immunoglobulin molecule in which the light chain is linked to the heavy chain by a disulfide bond). Polypeptides may have or form one or more intrachain disulfide bonds. With respect to the polypeptides described herein, reference to amino acid residues as defined by SEQ ID NOs includes post-translational modifications of such residues. In the context of this document, the expression CD3-binding protein can be used interchangeably with CD3-binding polypeptide. Such molecules bind specifically to the cassette of differentiation 3 (CD3) protein (eg, human CD3).

A protein is a macromolecule containing one or more polypeptide chains. The protein may also contain non-peptide components such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to the protein by the cell in which the protein is made and vary by cell type. In this document, proteins are defined in terms of their amino acid backbone structures; substituents, such as carbohydrate groups, are generally not specified, but may nonetheless be present. The protein may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, the protein may also be an scFv-Fc-scFv molecule, an scFv-scFv dimer, or a diabody.

The terms amino-terminal and carboxy-terminal are used herein to denote positions in polypeptides. Where the context permits, these terms are used in relation to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, some sequence located in the polypeptide carboxy-terminal to the standard sequence is located near the carboxy-terminus of the standard sequence, but not necessarily at the carboxy-terminus of the entire polypeptide. The T cell receptor (TCR) is a T cell surface molecule that, along with CD3, is generally responsible for the recognition of antigens associated with major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of highly variable a and β chains in most T cells. Other T cells express an alternative receptor consisting of variable γ and δ chains. Each TCR chain is a member of the immunoglobulin superfamily and contains one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus (see Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor : Saunders, Philadelphia, 2003; Janeway et al., Immunobiology: The Immune System in Health and Disease, ^4th Ed., Current Biology Publications, pp. 148, 149 and 172, 1999). Used in the present invention, the TCR can be obtained from various animal species, including humans, mice, rats or other mammals.

As used herein, a TCR complex refers to a complex formed by association of CD3 chains with other TCR chains. For example, a TKR complex may consist of a CD3y chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3Z chains, a TKPa chain, and a TKRb chain. Alternatively, the TKR complex may consist of a CD3y chain, a CD3δ chain, two CD3ε homodimer chains from the CD3Z chains of the ΤΚΡγ chain and a TKRδ chain.

As used herein, a component of a TCR complex refers to a TCR chain (i.e., TKR a, ΤΚΡβ, ΤΚΡγ, or ΤΚΡδ), a CD3 chain (i.e., CD3y, CD3δ, CD3ε, or CD3Z, or a complex formed by two or more TCR chains or chains CD3 (e.g., TKPa and ΤΚΡβ complex, ΤΚΡγ and ΤΚΡδ complex, CD3ε and CD3δ complex, CD3y and CD3ε complex, or sub-TKR complex of TKPa, ΤΚΡβ, CD3y, CD3δ, and two CD3ε chains).In the context of this document, antibody-dependent cell-mediated cytotoxicity and ADCC refers to a cell-mediated process in which non-specific cytotoxic cells that express FcyR (e.g., monocytes such as natural killer (NK) cells and macrophages) recognize a bound antibody (or other protein capable of binding FcyR) on a target cell and subsequently lead to lysis of the target cell In principle, any effector cell with an activating FcyR can be stimulated to mediate ADCC The main cells to mediate ADCC are NK cells and, which express only FcyRIII, while monocytes, depending on their state of activation, localization, or differentiation, may express FcyRI, FcyRII, and FcyRIII. Overview of FcyR expression. in hematopoietic cells, see, for example, Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92.

The term having ADCC activity, as used herein in relation to a polypeptide or protein, means that the polypeptide or protein (for example, containing an immunoglobulin hinge region and an immunoglobulin constant region containing CH2 and CH3 domains, for example derived from IgG (for example, IgG1)) capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) by binding to a cytolytic Fc receptor (eg FcyRIII) on a cytolytic immune effector cell expressing the Fc receptor (eg NK cell).

In the context of this document, complement-dependent cytotoxicity and CSC refers to the process in which components in normal serum (complement), together with an antibody or other Clq-complement-binding protein bound to a target cell, exhibit lysis of a target cell expressing the target antigen. Complement consists of a group of serum proteins that act together and in sequence to perform their function. In the context of this document, the terms classical complement pathway and classical complement system are synonymous and refer to a specific complement activation pathway. The classical pathway requires antigen-antibody complexes to initiate and involves the activation, in order, of nine major protein components, designated C1 to C9. The product of several stages of the activation process is an enzyme that catalyzes the next stage. This cascade allows for the amplification and activation of large amounts of complement through a relatively small initial signal. The term having CSC activity, as used herein in relation to a polypeptide or protein, means that a polypeptide or protein (for example, containing an immunoglobulin hinge region and an immunoglobulin constant region containing CH2 and CH3 domains, for example, derived from IgG (for example, IgG1) ) is able to mediate complement-dependent cytotoxicity (CDC) by binding the complement protein C1q and activating the classical complement system.

In the context of this document, redirected T cell cytotoxicity and PTCC refers to a T cell mediated process in which a cytotoxic T cell is recruited to a target cell by a multispecific protein that is able to specifically bind both the cytotoxic T cell and the target cell. , and in doing so, a target-dependent T-cell response occurs against the target cell. The CD3-binding domain-containing polypeptides and proteins and tumor antigen-binding domains described herein are capable of PTCC. In the context of this document, the terms treatment or reduction of intensity refers to therapeutic treatment or prophylactic/preventive treatment. The treatment is therapeutic if there is an improvement in at least one symptom of the disease in the individual being treated, or the treatment can slow the worsening of a progressive disease in the individual or prevent the occurrence of additional associated diseases.

As used herein, the term "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a particular binding molecule or compound refers to an amount of a compound sufficient to reduce the intensity of one or more symptoms of the disease being treated in a statistically significant manner or statistically significant improvement. organ function. When referring to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When it comes to combination, a therapeutically effective dose refers to the combined amounts of active ingredients that result in a therapeutic effect when administered sequentially or simultaneously (in the same formulation or in parallel in different formulations).

In the context of this document, the terms transformation, transfection, and transduction refer to the transfer of a nucleic acid (ie, nucleotide polymer) into a cell. As used herein, the term genetic transformation refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The transferred nucleic acid can be introduced into the cell using an expression vector.

In the context of this document, the term variant or variants refers to a nucleic acid or polypeptide that differs from the standard nucleic acid or polypeptide, but retains their important properties. In general, variants are very similar and in many areas identical to a standard nucleic acid or polypeptide. For example, a variant may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity compared to the active portion of a full-length reference nucleic acid or polypeptide.

- 15 042409

The terms light chain variable region (also referred to as light chain variable domain or VL or VL) and heavy chain variable region (also referred to as heavy chain variable domain or VH or VH) refer to the variable binding region of an antibody light and heavy chain, respectively. Variable binding regions consist of discrete, well-defined subregions known as complementarity determining regions (CDRs) and framework regions (FRs), generally arranged in the order FR1-CDR1-FR2-CDR2-FR3CDR3-FR4 from amino-terminus to carboxy-terminus. end. In one embodiment, the FRs are humanized. The term CL refers to an immunoglobulin light chain constant region or a light chain constant region, i.e. constant region from the light chain of the antibody. The term CH refers to an immunoglobulin heavy chain constant region or a heavy chain constant region that is further divided, depending on the antibody isotype, into CH1, CH2, and CH3 (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 (IgE, IgM). The Fab (antigen binding fragment) is the portion of an antibody that binds to antigens and contains the variable region and heavy chain CH1 domain linked to the light chain by an interchain disulfide bond.

The present invention describes binding domains that specifically bind CD3 (eg, human CD3), as well as polypeptides and proteins containing these binding domains. In some embodiments, CD3 binding proteins and polypeptides contain a second binding domain that can bind to a tumor antigen (e.g., PSMA, CD19, CD20, CD37, CD38, CD123, Her2, ROR1, RON, A33 glycoprotein antigen (gpA33), or CEA ). The polypeptides and proteins containing the binding domains of the present invention may further comprise immunoglobulin constant regions, linker peptides, hinge regions, immunoglobulin dimerization/heterodimerization domains, junction amino acids, labels, and the like. These components of the disclosed polypeptides and proteins are described in more detail below. In addition, the CD3 binding proteins and polypeptides described herein may be in the form of an antibody or fusion protein in any of a variety of different formats (eg, the fusion protein may be in the form of a CD3 binding bispecific or multispecific molecule). Non-limiting examples of bispecific molecules include the scFv-Fc-scFv molecule. Some bispecific molecules typically contain or consist of an anti-tumor antigen scFv linked to an anti-CD3 scFv and typically do not contain other sequences such as an immunoglobulin constant region. In other embodiments, the CD3 binding protein is a diabody.

The CD3 binding protein of the present invention generally comprises at least one CD3 binding polypeptide chain comprising (a) the CD3 binding domain set forth herein. In certain variations, the CD3 binding polypeptide further comprises (b) a hinge region located carboxy-terminally to the CD3 binding domain and (c) an immunoglobulin constant region. In further variations, the ROR1 binding polypeptide further comprises (d) a carboxy-terminal linker located carboxy-terminal to the immunoglobulin constant region and (e) a second binding domain located carboxy-terminal to the carboxy-terminal linker.

In other variations, the CD3 binding polypeptide comprises (b) a hinge region located amino-terminally to the CD3 binding domain, and (c) an immunoglobulin subregion located amino-terminally to the hinge region. In some variations, the CD3 binding polypeptide comprises (b) a hinge region located carboxy-terminal to the CD3 binding domain, and (c) an immunoglobulin subregion located carboxy-terminal to the hinge region. In some embodiments, CD3 binding polypeptides are capable of homodimerization, typically by disulfide linkage, via an immunoglobulin constant region and/or hinge region (e.g., via an immunoglobulin constant region containing IgG CH2 and CH3 domains and an IgG hinge region). Thus, in certain embodiments of the present invention, two identical single chain CD3 binding polypeptides are homodimerized to form a dimeric CD3 binding protein. In other embodiments, the CD3 binding polypeptide comprises a heterodimerization domain capable of heterodimerization with a different heterodimerization domain in a second, non-identical polypeptide chain. In certain variations, the second polypeptide chain for heterodimerization contains a second binding domain. Accordingly, in certain embodiments of the present invention, two non-identical polypeptide chains, one containing a CD3 binding domain and the other optionally containing a second binding domain, dimerize to form a heterodimeric CD3 binding protein. Examples of types of heterodimers include those described in US 2013/0095097 and US 2013/0129723.

In some embodiments, the CD3 binding domain, protein, or polypeptide is conjugated to a drug or toxic compound. The CD3 binding polypeptides, proteins and various components thereof useful in the present invention are further described below.

As stated above, this invention relates to binding domains that specifically bind CD3 (eg, human CD3). CD3-binding domain may contain the amino acid sequences shown in table. 14. In some embodiments of CD3 binding

- 16 042409 a polypeptide or protein contains a signal sequence. This invention also includes CD3-binding domains and proteins containing or encoded by any of the sequences shown in table. 14 except for the signal sequences that are part of those sequences. CD3-binding domains and polypeptides, their internal designations and their sequences are summarized in table. 15. In some cases, the CD3 binding domains of the invention contain amino acid substitutions. For example, TSC370 has the amino acid sequence TSC342 in which the glycine residue at position 27, according to the IMGT numbering system, is replaced by tyrosine. In certain embodiments, the invention provides a CD3 binding domain that specifically binds to human CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is (a) at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical or at least about 99% identical to the amino acid sequence in SEQ ID NO: 88; or (b) at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 89; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 86. A CD3 binding domain may comprise an amino acid sequence that is at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least at least about 97% identical, at least about 98% identical, or at least approximately 99% identical to the amino acid sequence in SEQ ID NO: 83 or SEQ ID NO: 84. The CD3 binding domain may contain or consist of SEQ ID NO: 83 or SEQ ID NO: 84. In some embodiments, the CD3 binding domain contains an amino acid sequence that is at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60.

In some embodiments, the CD3 binding domain may comprise an immunoglobulin light chain variable region that contains an LCDR1 amino acid sequence that differs from SEQ ID NO: 94 by at least one amino acid substitution, an LCDR2 amino acid sequence that differs from SEQ ID NO: 95 by by at least one amino acid substitution, and an LCDR3 amino acid sequence that differs from SEQ ID NO: 96 by at least one amino acid substitution, and an immunoglobulin heavy chain variable region that contains an HCDR1 amino acid sequence that differs from SEQ ID NO: 91 by at least by one amino acid substitution, an HCDR2 amino acid sequence that differs from SEQ ID NO: 92 by at least one amino acid substitution, and an HCDR3 amino acid sequence that differs from SEQ ID NO: 93 by at least one amino acid substitution. In other embodiments, the CD3 binding domain may comprise an immunoglobulin light chain variable region that contains an LCDR1 amino acid sequence that differs from SEQ ID NO: 202 by at least one amino acid substitution, an LCDR2 amino acid sequence that differs from SEQ ID NO: 203 by by at least one amino acid substitution, and an LCDR3 amino acid sequence that differs from SEQ ID NO: 204 by at least one amino acid substitution, and an immunoglobulin heavy chain variable region that contains an HCDR1 amino acid sequence that differs from SEQ ID NO: 199 by at least by one amino acid substitution, an HCDR2 amino acid sequence that differs from SEQ ID NO: 200 by at least one amino acid substitution, and an HCDR3 amino acid sequence that differs from SEQ ID NO: 201 by at least one amino acid substitution. The amino acid sequence of a CD3 binding domain CDR may differ from said sequence by at least one amino acid substitution. The at least one amino acid substitution may be a conservative or non-conservative amino acid substitution. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3 are different from the above CDR sequences

- 17 042409 for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, a CDR of the present invention comprises about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) inserts, about one or more (e.g., about 2, 3, 4 , 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions) or a combination of the above changes compared to the CDR sequence of a known monoclinal antibody.

When describing amino acid substitutions in this invention, the position of an amino acid residue in the variable region of an immunoglobulin molecule is usually numbered using the IMGT system (Brocket X. et al., Nucl. Acids Res. (2008) 36, W503-508). In some embodiments, the amino acid residue at position 52 of the immunoglobulin variable light chain region of the CD3 binding domain is arginine and/or the amino acid residue at position 53 of the immunoglobulin variable light chain region of the CD3 binding domain is tryptophan. The amino acid residue at position 27 of the immunoglobulin heavy chain variable region of the CD3 binding domain may be tyrosine. In some embodiments, the CD3 binding domain comprises one or more of the following: (a) the amino acid residue at position 9 of the immunoglobulin heavy chain variable region is a proline; (b) the amino acid residue at position 53 of the immunoglobulin heavy chain variable region is isoleucine; and (c) the amino acid residue at position 21 of the immunoglobulin light chain variable region is methionine. The amino acid residue at position 87 of the immunoglobulin light chain variable region of the CD3 binding domain may be tyrosine. The amino acid residue at position 86 of the immunoglobulin heavy chain variable region of the CD3 binding domain may be aspartic acid. In one embodiment, the amino acid residue at position 86 of the CD3 binding domain immunoglobulin light chain variable region is aspartic acid and the amino acid residue at position 87 of the CD3 immunoglobulin light chain variable region is tyrosine. In certain embodiments, the CD3 binding domain comprises humanized VL and/or VH regions of an immunoglobulin. Methods for humanizing the VL and VH regions of an immunoglobulin are known in the art and are discussed, for example, in US Patent Application Publication No. 2006/0153837. In certain aspects, a CD3 binding domain may comprise an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region that comprise framework regions, and at least one of an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region may be humanized. In one embodiment, the immunoglobulin light chain variable region comprises framework regions based on the human germline amino acid sequence IGKV3D-20*1. In another embodiment, the immunoglobulin heavy chain variable region comprises framework regions based on the amino acid sequence of the human germline IGHV1-69*02. In some aspects, the immunoglobulin heavy chain variable region comprises framework regions based on the human germline amino acid sequence IGHV1-2*02 (H7), IGHV1-46*02 (H8), IGHV1-3*01(H9), or IGHV1-69*02 (H10). An immunoglobulin light chain variable region may comprise framework regions based on the human germline amino acid sequence IGKV3-11*01 (L4), IGKV1-33*01 (L5), IGKV1-39*01 (L7), or IGKV3D-20*1 (L8). ).

This invention relates to CD3 binding domains that have improved properties compared to the CD3 binding domain of DRA222. DRA222 has a light chain variable region containing SEQ ID NO: 90 and a heavy chain variable region containing SEQ ID NO: 87. DRA222 is described in WO 2013/158856. In this invention, DRA222 is sometimes referred to as TSC311 or TSC312. Fc DRA222 has the amino acid sequence of SEQ ID NO: 2. This invention includes a CD3 binding domain (or a protein containing the specified domain), which has a thermal stability increased by at least 10% compared with the thermal stability of the CD3 binding domain containing the variable region a light chain comprising SEQ ID NO: 90; and a heavy chain variable region comprising SEQ ID NO: 87. °C, at least about 4°C, at least about 5°C, or at least about 6°C and up to about 20°C compared to the Tm of the CD3-binding domain containing the light chain variable region, containing SEQ ID NO: 90, and a heavy chain variable region containing SEQ ID NO: 87. at least about 54°C, at least about 55°C, at least about 56°C, or at least about 57°C, and up to about 72°C. The thermal stability or midpoint of the thermal transition of a CD3 binding domain (or a protein containing said domain) can be determined using differential scanning calorimetry or differential scanning fluorimetry.

- 18 042409

A CD3 binding domain (or a protein comprising said domain) described herein may have a storage stability increased by at least about 5%, at least about 10%, at least about 20%, at least by about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% and up to approximately 100% compared to the storage stability of a CD3 binding domain containing a light chain variable region containing SEQ ID NO: 90 and a heavy chain variable region containing SEQ ID NO: 87. Storage stability can be determined after storing the CD3 binding domain (or a protein containing said domain) in PBS at approximately 25°C. In one embodiment, the CD3 binding domain (or protein containing said domain) is stable when stored in PBS at about 25° C. for at least about 6 days, at least about 10 days, or at least about 13 days and up to approximately 90 days.

In some aspects, a CD3 binding domain (or a protein comprising said domain) described herein has a level of high molecular weight aggregates formed during recombinant expression that is at least about 5%, at least about 10%, at least about 20% reduced, at least about 30% reduced, and up to about 50% reduced compared to the level of high molecular weight aggregates formed during recombinant expression of a CD3 binding domain containing a light chain variable region containing SEQ ID NO: 90, and a heavy chain variable region comprising SEQ ID NO: 87. The invention also relates to a CD3 binding domain that binds to human CD3 with an EC ₅₀ of about 10 nM or less. The CD3 binding domain of the invention can specifically bind to both human CD3 and cynomolgus CD3. For example, a CD3 binding domain can bind to cynomolgus monkey CD3 with an EC ₅₀ of about 30 nM or less. Binding to cynomolgus monkey CD3 allows testing of anti-CD3 therapeutics in non-human primates. The present invention includes a CD3 binding domain that specifically binds to human CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region comprises an LCDR1 amino acid sequence according to SEQ ID NO: 94, an LCDR2 amino acid sequence according to SEQ ID NO: 95 and the amino acid sequence of LCDR3 according to SEQ ID NO: 96, and the immunoglobulin heavy chain variable region contains the amino acid sequence of HCDR1 according to SEQ ID NO: 91, the amino acid sequence of HCDR2 according to SEQ ID NO: 92 and the amino acid sequence of HCDR3 according to SEQ ID NO: 93; and wherein the CD3 binding domain has one or more of the properties described herein. For example, (i) the midpoint of the thermal transition of the CD3 binding domain (or protein containing the CD3 binding domain) is at least about 54°C, at least about 55°C, at least about 56°C, or at least about 57°C and up to about 72°C; (ii) the CD3 binding domain (or protein containing the CD3 binding domain) is stable when stored in PBS at about 25° C. for at least about 6 days, at least about 10 days, or at least about 13 days, and up to approximately 90 days; (iii) a CD3 binding domain (or a protein containing a CD3 binding domain) binds to human CD3 with an EC ₅₀ of about 10 nM or less; and (iv) a CD3 binding domain (or a protein containing a CD3 binding domain) binds to cynomolgus monkey CD3 with an EC ₅₀ of about 30 nM or less.

In some embodiments, a CD3 binding polypeptide, when bound to a CD3 protein on a T cell, induces no or minimal detectable release of cytokines from said T cell. In certain aspects, the CD3 binding protein or polypeptide exhibits reduced cytokine release in the patient compared to cytokine release when an anti-CD3 OCTP antibody is administered to the patient. A CD3 binding polypeptide can induce T cell activation or T cell proliferation.

In certain embodiments, the CD3 binding protein may comprise one or more additional binding domains (eg, a second binding domain) that bind to a target other than CD3. These other binding domains may include, for example, a particular cytokine or molecule that targets the polypeptide's binding domain to a particular cell type, toxin, additional cell receptor, antibody, and so on.

In certain embodiments, the CD3 binding polypeptide used with the methods and compositions described herein is a bispecific single chain molecule containing a CD3 binding domain and a second binding domain. In some embodiments, the CD3 and/or second binding domain is derived from an antibody and contains a variable heavy chain (VH) and a variable light chain (VL). These binding domains and variable chains

- 19 042409 may be arranged in any order that retains some degree of binding to the target(s). For example, the variable domains may be in the order VH VVD-VL VVD-VH CD3-VL CD3; VL VSD-VH VSD-VH CD3-VL CD3; VH VSD-VL VSD-VL CD3-VH CD3; VL VSD-VH VSD-VL CD3-VH CD3; VH CD3-VL CD3-VH VSD-VL VSD; VL CD3-VH CD3-VL VSD-VH VSD; VH CD3-VL CD3-VL VSD-VH VSD; or VL CD3-VH CD3-VH VSD-VL VVD (where VVD refers to the second binding domain). In certain aspects, the pairs of VH and VL regions in the CD3 binding domain are presented in single chain antibody (scFv) format. The VH and VL regions may be arranged in the order VH-VL or VL-VH. In certain embodiments, the scFv may more efficiently bind to CD3 in the VL-VH orientation than in the VH-VL orientation, or vice versa. The VH region may be located N-terminally to the linker sequence. The VL region may be located C-terminally to the linker sequence. The location of the domains in the CD3 binding domain of the bispecific single chain molecule may be VH-VL, with said CD3 binding domain located C-terminally to the second binding domain. In some cases, the bispecific molecule may contain an scFv that binds to a second binding domain associated with the scFv that binds to CD3. These scFvs can be linked by a short peptide. In some embodiments, bispecific single chain molecules do not contain a hinge region or a constant region (see, for example, US 2013/0295121, US 2013/0129730, US 2011/0293619, US 7635472, WO 2010/037836, WO 2004/106381 and WO 2011 /121110; incorporated herein by reference in its entirety).

In some embodiments, the binding domain is a single chain Fv fragment (scFv) that contains V _H and VL regions specific to the target of interest. In certain embodiments, the V _H and V _L regions are human or humanized. In some variations, the binding domain is a single chain Fv (scFv) containing the V _H and V _L regions of an immunoglobulin connected by a peptide linker. The use of peptide linkers to connect the VH and VL regions is well known in the art and there are a large number of publications in this particular area. The linker may have the amino acid sequence QRHNNSSLNTGTQMAGHSPNS (SEQ ID NO: 148). In some embodiments, the peptide linker is a 15-mer consisting of three repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser ((Gly ₄ Ser) ₃ ) (SEQ ID NO: 193). Other linkers have also been used, and phage display as well as selective infection phage assays have been used to expand diversity and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). In certain embodiments, the VH and VL regions are connected by a peptide linker having the amino acid sequence of the formula (Gly ₄ Ser) _n , where n = 1-5 (SEQ ID NO: 194). In some embodiments, the Gly ₄ Ser sequence may be repeated 6-10 times. Other suitable linkers can be obtained by optimizing a simple linker (eg, (Gly ₄ Ser) _n (SEQ ID NO: 194)) through random mutagenesis. In some embodiments, the scFv heavy chain variable region is amino-terminal to the scFv light chain variable region. In other embodiments, the scFv light chain variable region is amino-terminal to the scFv heavy chain variable region.

In some embodiments, the CD3 binding polypeptide comprises, in amino-terminal to carboxy-terminal order (or in carboxy-terminal to amino-terminal order), (i) a second binding domain, (ii) a hinge region, (iii) a constant region immunoglobulin, (iv) a carboxy-terminal linker (or amino-terminal linker), and (v) a CD3 binding domain. As used herein in the context of a polypeptide construct comprising a first binding domain and a second binding domain, a hinge region or hinge refers to the polypeptide region between the first binding domain and the Fc region. Carboxy-terminal linker or amino-terminal linker refers to the polypeptide region between the Fc region and the second binding domain. In some embodiments, the carboxy-terminal (or amino-terminal) linker comprises or consists of SEQ ID NO: 196. In certain embodiments, the hinge region is a wild-type human immunoglobulin hinge region. In certain other embodiments, amino acid residues may be added to the amino or carboxy terminus of the hinge region of a human wild-type immunoglobulin as part of a fusion protein construct. For example, additional connecting amino acid residues at the amino end of the hinge may be RT, RSS, TG, or T, or at the carboxy end of the hinge may be SG, or a deletion in the hinge may be combined with an addition, such as AP with the addition SG at the carboxy end. In certain embodiments, the hinge region, carboxy-terminal linker, or amino-terminal linker is an altered immunoglobulin hinge region in which one or more cysteine residues in the wild-type immunoglobulin hinge region are replaced with one or more other amino acid residues (e.g., serine or alanine) .

Exemplary altered immunoglobulin hinge regions, carboxy-terminal linkers, and amino-terminal linkers include a human IgG1 immunoglobulin hinge region in which one, two, or three cysteine residues present in wild-type human IgG1 hinge region

- 20 042409 type, substituted with one, two or three different amino acid residues (eg serine or alanine). In the altered hinge region of the immunoglobulin, proline substitution with another amino acid (eg, serine or alanine) may additionally be present. For example, in the altered human IgG1 hinge region described above, there may additionally be a replacement of the proline located carboxy-terminally to the three cysteines of the wild-type human IgG1 hinge region with another amino acid residue (eg, serine, alanine). In one embodiment, the prolines in the central part of the hinge region remain unsubstituted. In certain embodiments, the hinge, carboxy-terminal linker, or amino-terminal linker polypeptide contains or is a sequence that is at least 80%, at least 81%, at least 82%, at least 83% for at least

84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least

92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98%, or at least 99% identical to a wild-type immunoglobulin hinge, e.g., wild-type human IgG1 hinge, wild-type human IgG2 hinge, or human IgG4 hinge wild type.

In additional embodiments, the hinge, carboxy-terminal linker, or amino-terminal linker present in the CD3 binding polypeptide may be a hinge region that is not based on or derived from an immunoglobulin hinge region (i.e., is not hinge region of wild-type immunoglobulin or altered hinge region). Examples of such hinge regions and carboxy-terminal linkers include peptides of about five to about 150 amino acids derived from the transmembrane protein interdomain or stem region of a type II C lectin, such as peptides of about eight to 25 amino acids, and peptides , consisting of approximately seven to 18 amino acids.

In certain embodiments, interdomain or stem-derived hinges, carboxy-terminal linkers, and amino-terminal linkers have seven to 18 amino acids and can form an α-supercoil structure. In certain embodiments, the interdomain or stem-derived hinge carboxy-terminal linkers or amino-terminal linkers contain 0, 1, 2, 3, or 4 cysteine. Exemplary interdomain or stem-derived hinge regions, carboxy-terminal linkers, and amino-terminal linkers are peptide fragments of the interdomain or stem regions, such as 150 amino acid fragments from the stem regions of CD69, CD72, CD94, NKG2A, and NKG2D. The hinge region, carboxy-terminal linker or amino-terminal linker can also be a flexible linker sequence containing repeats (Gly ₄ Ser). In some embodiments, the hinge region is a 15-mer consisting of three repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser ((Gly ₄ Ser) ₃ ) (SEQ ID NO: 193). In certain embodiments, the hinge region has an amino acid sequence of the formula (Gly ₄ Ser) _n where n = 1-5 (SEQ ID NO: 194). In some embodiments, the Gly ₄ Ser sequence may be repeated 6-10 times. Other suitable hinge regions can be obtained by optimizing a simple linker (eg, (Gly ₄ Ser) _n (SEQ ID NO: 194)) by random mutagenesis.

In certain embodiments, the hinge, carboxy-terminal linker, and amino-terminal linker sequences comprise from about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, from 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids. The hinge region or linker may be predominantly flexible, but may also provide more rigid characteristics, or may contain a predominantly α-helical structure with a minimum of β-sheet structure. The lengths or sequences of the hinge regions and linkers can affect the binding affinity of the binding domains to which the hinge regions are directly or indirectly (via another region or domain, such as a heterodimerization domain) connected, as well as one or more of the activities of the portions of the Fc region, with by which the hinge regions or linkers are directly or indirectly connected.

In certain embodiments, the hinge, carboxy-terminal linker, and amino-terminal linker sequences are plasma and serum stable and resistant to proteolytic cleavage. The first lysine in the upper hinge region of IgG1 may be mutated to minimize proteolytic cleavage, for example the lysine may be substituted with methionine, threonine, alanine or glycine, or removed.

In some embodiments, the CD3 binding protein is capable of forming a heterodimer with a second polypeptide chain and contains a hinge region (a) located directly amino-terminally to an immunoglobulin constant region (e.g., amino-termi

- 21 042409 directly to the CH2 domain, with the immunoglobulin constant region containing the CH2 and CH3 domains, or amino-terminally to the CH3 domain, with the immunoglobulin subregions containing the CH3 and CH4 domains), (b) inserted between and connecting the binding domain (for example, scFv) and an immunoglobulin heterodimerization domain, (c) inserted between and connecting the immunoglobulin heterodimerization domain and the immunoglobulin constant region (e.g., when the immunoglobulin constant region contains CH2 and CH3 domains or CH3 and CH4 domains), (d) inserted between and connecting the immunoglobulin constant region and the binding domain, (e) at the amino terminus of the polypeptide chain or (f) at the carboxy terminus of the polypeptide chain. A polypeptide chain containing the hinge region described herein is capable of binding to another polypeptide chain to form a heterodimeric protein as provided herein, and the resulting heterodimer will contain a binding domain that retains the target specificity or specific binding affinity of the target. In certain embodiments, a hinge region present in a polypeptide that forms a heterodimer with another polypeptide chain may be an immunoglobulin hinge region, such as a wild-type immunoglobulin hinge region or an altered immunoglobulin hinge region. In certain embodiments, the hinge region of one of the polypeptide chains of the heterodimeric protein is identical to the corresponding hinge region of the other polypeptide chain of the heterodimer. In certain other embodiments, the hinge region of one of the chains differs from the hinge region of the other chain (in length or sequence). The different hinge regions in different chains allow for different manipulations of the binding affinity of the binding domains to which the hinge regions are linked such that the heterodimer is able to preferentially bind to the target of one binding domain over the target of another binding domain. For example, in certain embodiments, the heterodimeric protein contains a CD3 binding domain on one strand and a second binding domain on the other strand. The presence of two different hinge regions in the two chains may allow the heterodimer to bind first to the second target and then to the CD3 component of the first. Thus, the heterodimer can recruit CD3+ T cells to cells expressing the second target (eg, tumor or cancer cells), and they in turn can damage or destroy cells expressing the second target.

Some examples of hinge region, carboxy-terminal linker and amino-terminal linker sequences suitable for use in accordance with the present invention are shown in Table 1 below. 1 and 2. Additional examples of hinge and linker regions are shown in SEQ ID NOs: 241-244, 601, 78, 763-791, 228, 379-434, 618-749 in US 2013/0129723 (these sequences are incorporated herein by links).

- 22 042409

Table 1. Examples of hinge regions and linkers

Hinge area Amino acid sequence SEQID NO sss(s)-hlgG1 hinge EPKSSDKTHTSPPSS SEQ ID NO:121 csc(s)-hlgG1 hinge EPKSCDKTHTSPPCS SEQ ID NO:122 ssc(s)-hlgG1 hinge EPKSSDKTHTSPPCS SEQ ID NO:123 scc(s)-hlgG1 hinge EPKSSDKTHTCPPCS SEQ ID NO:124 css(s)-hlgG1 hinge EPKSCDKTHTSPPSS SEQ ID NO:125 scs(s)-hlgG1 hinge EPKSSDKTHTCPPSS SEQ ID NO:126 ccc(s)-hlgG1 hinge EPKSCDKTHTSPPCS SEQ ID NO:127 ccc(p)-hlgG1 hinge EPKSCDKTHTSPPCP SEQ ID NO:128 sss(p)-hlgG1 hinge EPKSSDKTHTSPPSP SEQ ID NO:129 csc(p)-hlgG1 hinge EPKSCDKTHTSPPCP SEQ ID NO:130 ssc(p)-hlgG1 hinge EPKSSDKTHTSPPCP SEQ ID NO:131 scc(p)-hlgG1 hinge EPKSSDKTHTCPPCP SEQ ID NO:132 css(p)-hlgG1 hinge EPKSCDKTHTSPPSP SEQ ID NO:133 scs(p)-hlgG1 hinge EPKSSDKTHTCPPSP SEQ ID NO:134 Scppcp SCPPCP SEQ ID NO:135 STD1 NYGGGGSGGGGSGGGGSGNS SEQ ID NO:136 STD2 NYGGGGSGGGGSGGGGSGNY GGGGSGGGGSGGGGSGNS SEQ ID NO:137 H1 NS SEQ ID NO:138 H2 GGGGSGNS SEQ ID NO:139 NZ NYGGGGSGNS SEQ ID NO:140 H4 GGGGSGGGGSGNS SEQ ID NO:141 H5 NYGGGGSGGGGSGNS SEQ ID NO:142 H6 GGGGSGGGGSGGGGSGNS SEQ ID NO:143 H7 GCPPCPNS SEQ ID NO:144 (G ₄ S) ₃ GGGGSGGGGSGGGGS SEQ ID NO:145 H105 SGGGGSGGGGSGGGGS SEQ ID NO:146 ( _G4S )4 GGGGSGGGGSGGGGSGGGGS SEQ ID NO:147 H75 (quadruple NKG2A mutant) QRHNNSSLNTGTQMAGHSPNS SEQ ID NO:148 H83 (derived from NKG2A) SSLNTGTQMAGHSPNS SEQ ID NO:149 H106 (derived from NKG2A) QRHNNSSLNTGTQMAGHS SEQ ID NO:150 H81 (derived from NKG2D) EVQIPLTESYSPNS SEQ ID NO:151 H91 (derived from NKG2D) NSLANQEVQIPLTESYSPNS SEQ ID NO:152 H94 SGGGGSGGGGSGGGGSPNS SEQ ID NO:153 H111 SGGGGSGGGGSGGGGSPGS SEQ ID NO:196

- 23 042409

Table 2. Examples of hinge regions and linkers (derived from H7 hinge region, type II lectin stem region C, or type I transmembrane protein interdomain region)

Hinge area Amino acid sequence Parent molecule and/or hinge region SEQID NO: H16 LSVKADFLTPSIGNS CD80 SEQ ID NO:154 H17 LSVKADFLTPSISCPPCPNS CD80 + H7 SEQ ID NO:155 H18 LSVLANFSQPEIGNS CD86 SEQ ID NO:156 H19 LSVLANFSQPEISCPPCPNS CD86 + H7 SEQ ID NO:157 H20 LKIQERVSKPKISNS CD2 SEQ ID NO:158 H21 LKIQERVSKPKISCPPCPNS CD2 + H7 SEQ ID NO:159 H22 LNVSERPFPPHIQNS CD22 SEQ ID NO:160 H23 LDVSERPFPPHIQSCPPCPNS CD22 + H7 SEQ ID NO:161 H24 REQLAEVTLSLKANS CD80 SEQ ID NO:162 H25 REQLAEVTLSLKACPPCPNS CD80 + H7 SEQ ID NO:163 H26 RIHQMNSELSVLANS CD86 SEQ ID NO:164 H27 RIHQMNSELSVLACPPCPNS CD86 + H7 SEQ ID NO:165 H28 DTKGKNVLEKIFSNS CD2 SEQ ID NO:166 NZO LPPETQESQEVTLNS CD22 SEQ ID NO:167 H32 RIHLNVSERPFPPNS CD22 SEQ ID NO:168 NZZ RIHLNVSERPFPPCPPCPNS CD22 + H7 SEQ ID NO:169 H36 GCPPCPGGGGSNS H7 SEQ ID NO:170 H40 GCPPCPANS H7 SEQ ID NO:171 H41 GCPPCPANS H7 SEQ ID NO:172 H42 GCPPCPNS H7 SEQ ID NO:173 H44 GGGASCPPPCPGNS H7 SEQ ID NO:174 H45 GGGASCPPCAGNS H7 SEQ ID NO:175 H46 GGGASCPPCANS H7 SEQ ID NO:176 H47 LSVKADFLTPSIGNS CD80 SEQ ID NO:177 H48 ADFLTPSIGNS CD80 SEQ ID NO:178 H50 LSVLANFSQPEIGNS CD86 SEQ ID NO:179 H51 LSVLANFSQPEIGNS CD86 SEQ ID NO:180 H52 SQPEIVPINSNS CD86 SEQ ID NO:181 H53 SQPEIVPISCPPCPNS CD86 + H7 SEQ ID NO:182 H54 SVLANFSQPEISCPPCPNS CD86 + H7 SEQ ID NO:183 H55 RIHQMNSELSVLANS CD86 SEQ ID NO:184 H56 QMNSELSVLANS CD86 SEQ ID NO:185 H57 VSERPFPPNS CD22 SEQ ID NO:186 H58 KPFFTCGSADTCPNS CD72 SEQ ID NO:187 H59 KPFFTCGSADTCPNS CD72 SEQ ID NO:188 H60 QYNCPGQYTFSMPNS CD69 SEQ ID NO:189 H61 EPAFTPGPNIELQKDSDCPNS CD94 SEQ ID NO:190 H62 QRHNNSSLNTRTQKARHCPNS NKG2A SEQ ID NO:191 H63 NSLFNQEVQIPLTESYCPNS NKG2D SEQ ID NO:192

In certain embodiments, a CD3 binding polypeptide or protein of the invention may comprise an immunoglobulin dimerization domain or an immunoglobulin heterodimerization domain.

As used herein, an immunoglobulin dimerization domain or an immunoglobulin heterodimerization domain refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts with or binds to a different immunoglobulin domain of another polypeptide chain, wherein the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or effectively promotes heterodimerization of the first and second polypeptide chains ( i.e. the formation of a dimer between two different polypeptide chains, which is also called a heterodimer or heterodimeric protein). The interaction between immunoglobulin heterodimerization domains contributes significantly to or effectively stimulates heterodimerization of the first and second polypeptide chains if there is a statistically significant decrease

dimerization between the first and second polypeptide chains in the absence of an immunoglobulin heterodimerization domain of the first polypeptide chain and/or an immunoglobulin heterodimerization domain of the second polypeptide chain. In certain embodiments, if the first and second polypeptide chains are expressed together, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least about 80 % to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the first and second polypeptide chains form heterodimers with each other. Exemplary immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL1 domain (e.g., Ck and Cλ isotypes), or derivatives thereof, including CH1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains such as provided by in this document. Dimerization/heterodimerization can be used if there is a need to form heterodimers from two non-identical polypeptide chains, one or both of the polypeptide chains containing a binding domain. In certain embodiments, one of the polypeptide chains of certain heterodimers described herein does not contain a binding domain. As stated above, the heterodimeric protein of the present invention contains an immunoglobulin heterodimerization domain in each polypeptide chain. The immunoglobulin heterodimerization domains of the polypeptide chains of the heterodimer differ from each other and therefore can be differently modified to facilitate heterodimerization of both chains and to minimize the homodimerization of each chain. The immunoglobulin heterodimerization domains provided herein allow efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric protein. As provided herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides (e.g., one short and one long) according to the present invention comprise immunoglobulin CH1 and CL domains, e.g., human CH1 and CL domains. . In certain embodiments, an immunoglobulin heterodimerization domain is a wild-type CH1 domain, such as the wild-type CH1 domain of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM. In further embodiments, the immunoglobulin heterodimerization domain is the wild-type CH1 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM as shown in SEQ ID NOs: 114, 186-192, and 194, respectively, in US 2013 /0129723 (these sequences are incorporated herein by reference). In certain embodiments, the immunoglobulin heterodimerization domain is the wild-type human IgG1 CH1 domain shown in SEQ ID NO: 114 in US 2013/0129723 (the sequence is incorporated herein by reference).

In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CH1 domain, such as an altered IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain. In certain embodiments, an immunoglobulin heterodimerization domain is an altered CH1 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM. In additional embodiments, the cysteine residue of the wild-type CH1 domain (e.g., human CH1) involved in disulfide bond formation with the immunoglobulin CL domain (e.g., human CL) is deleted or substituted in the altered immunoglobulin CH1 domain such that between the altered CH1 domain and CL the wild-type domain does not form a disulfide bond. In certain embodiments, the immunoglobulin heterodimerization domain is a wild-type CL domain, such as a wild-type Ck domain or a wild-type Cλ domain. In certain embodiments, an immunoglobulin heterodimerization domain is a human wild-type CK domain or a human wild-type Cλ domain, such as those shown in SEQ ID NOs: 112 and 113, respectively, in US 2013/0129723 (these sequences are incorporated herein by reference) . In further embodiments, the immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or Cλ domain, eg, an altered human CK or human Cλ domain.

In certain embodiments, a cysteine residue of a wild-type CL domain (eg, human CL) involved in disulfide bond formation with a wild-type immunoglobulin CH1 domain (eg, human CH1) is deleted or substituted in the altered immunoglobulin CL domain. Such altered CL domains may further comprise an amino acid deletion at the amino termini. An example of a Ck domain is shown in SEQ ID NO: 141 in US 2013/0129723 (the sequence is incorporated herein by reference), in which the first arginine and the last cysteine of the human wild type Ck domain are removed. In certain embodiments, only the last cysteine of the human wild-type CK domain is removed in the altered CK domain, since the first arginine removed from the human wild-type CK domain can be provided by a linker that contains arginine at the carboxy-terminus and links the amino-terminus an altered Ck domain with a different domain (e.g., an immunoglobulin subregion, such as the subregion containing the CH2 and CH3 domains of immunoglobulin) document by reference) in which the first arginine of the human wild-type Cλ domain is removed and the cysteine involved in the formation of a disulfide bond with the cysteine in the CH1 domain is replaced by serine.

In further embodiments, an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions from the wild-type CK domain at positions that may be involved in the formation of an interstrand hydrogen bonding network at the CK-SK interface. For example, in certain embodiments, an immunoglobulin heterodimerization domain is an altered human Skdomain in which one or more amino acids at positions N29, N30, Q52, V55, T56, S68, or T70 are replaced with another amino acid. Amino acid numbering is based on their positions in the altered human Cck sequence shown in SEQ ID NO: 141 in US 2013/0129723 (the sequence is incorporated herein by reference). In certain embodiments, an immunoglobulin heterodimerization domain is an altered human Ck domain containing one, two, three, or four amino acid substitutions at positions N29, N30, V55, or T70. The amino acid used as a substituent at the above positions may be an alanine or a bulky side chain amino acid residue such as arginine, tryptophan, tyrosine, glutamate, glutamine or lysine. Additional amino acid residues that can be used to replace the amino acid residues of the human wild-type CK sequence at the above positions (eg, N30) include aspartate, methionine, serine, and phenylalanine. Examples of altered human Ck domains are shown in SEQ ID NO: 142-178 in US 2013/0129723 (these sequences are incorporated herein by reference). Altered human Ck domains are those that facilitate heterodimerization with the CH1 domain but minimize homodimerization with the other Ck domain. Exemplary altered human Ck domains are shown in SEQ ID NOs: 160 (N29WV55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176(N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30D V55A T70E), 839 N30M V55A T70E), 840 (N30S V55A T70E) and 841 (N30F V55A T70E) in US 2013/0129723 (these sequences are incorporated herein by reference).

In certain embodiments, in addition to or as an alternative to the mutations in the Cdomains described herein, both immunoglobulin heterodimerization domains (i.e., immunoglobulin CH1 and CL domains) of the polypeptide heterodimer contain mutations such that the resulting immunoglobulin heterodimerization domains form salt bridges. (i.e., ionic interactions) between amino acid residues in mutated regions. For example, the immunoglobulin heterodimerization domains of a polypeptide heterodimer can be a mutated CH1 domain in combination with a mutated Ck domain. In the mutated CH1 domain, the valine at position 68 (V68) of the wild-type human CH1 domain is replaced by a negatively charged amino acid residue (e.g., aspartate or glutamate), while the leucine at position 29 (L29) of the mutated human Ck domain, in which the first arginine and the last cysteine have been removed, replaced by an amino acid residue having a positive charge (for example, lysine, arginine, or histidine). A charge-charge interaction between an amino acid residue having a negative charge of the resulting mutated CH1 domain and an amino acid residue having a positive charge of the resulting mutated Ck domain results in the formation of a salt bridge that stabilizes the heterodimeric interface between the mutated CH1- and Sk-domains. Alternatively, wild-type V68 CH1 can be substituted with a positively charged amino acid residue, while L29 of a mutated human Ck domain in which the first arginine and last cysteine have been removed can be substituted with a negatively charged amino acid residue. Examples of mutated CH1 sequences in which V68 is substituted with a negative or positive amino acid residue are shown in SEQ ID NOs: 844 and 845 in US 2013/0129723 (these sequences are incorporated herein by reference). Examples of mutated Ck sequences in which L29 is substituted with a negative or positive amino acid residue are shown in SEQ ID NOs: 842 and 843 in US 2013/0129723 (these sequences are incorporated herein by reference).

Positions other than V68 of the human CH1 domain and L29 of the human Kk domain can be replaced with amino acids having opposite charges to provide ionic interactions between amino acids in addition to or as an alternative to mutations in V68 of the CH1 domain and L29 of the Kk domain. Such positions can be determined by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH1-CK pair, to determine amino acid residues at the CH1-CK interface, and further determine suitable positions among amino acid residues at the CH1-CK interface using a number of criteria (for example, predisposition to participate in ionic interactions, proximity to a potential partner residue, etc.).

In certain embodiments, the polypeptide heterodimers of the present invention contain only one pair of immunoglobulin heterodimerization domains. For example, the first chain of the polypeptide heterodimer may contain a CH1 domain as an immunoglobulin heterodimerization domain, while the second chain may contain a CL domain (eg, Ck or Cλ) as an immunoglobulin heterodimerization domain. Alternatively, the first strand may contain a CL domain (eg, Ck or Cλ) as an immunoglobulin heterodimerization domain, while the second strand may contain a CH1 domain as an immunoglobulin heterodimerization domain. As stated herein, the first and second chain immunoglobulin heterodimerization domains are capable of binding to form a heterodimeric protein of the invention. In certain other embodiments, the heterodimeric proteins of the present invention may comprise two pairs of immunoglobulin heterodimerization domains. For example, the first strand of the heterodimer may contain two CH1 domains, while the second strand may contain two CL domains that bind to two CH1 domains in the first strand. Alternatively, the first strand may contain two CL domains, while the second strand may contain two CH1 domains that bind to two CL domains in the first strand. In certain embodiments, the first polypeptide chain contains a CH1 domain and a CL domain, while the second polypeptide chain contains a CL domain and a CH1 domain that bind to the CH1 domain and CL domain, respectively, of the first polypeptide chain. In embodiments in which the heterodimeric protein contains only one heterodimerization pair (i.e., one immunoglobulin heterodimerization domain in each chain), the immunoglobulin heterodimerization domain of each chain may be located amino-terminally to the immunoglobulin constant region of that chain. Alternatively, the immunoglobulin heterodimerization domain in each chain may be located carboxy-terminally to the immunoglobulin constant region of that chain.

In embodiments in which the heterodimeric protein contains two pairs of heterodimerizations (i.e., two immunoglobulin heterodimerization domains in each chain), both immunoglobulin heterodimerization domains in each chain can be located amino-terminally to the immunoglobulin constant region of that chain. Alternatively, both immunoglobulin heterodimerization domains in each chain may be located carboxy-terminally to the immunoglobulin constant region of that chain. In additional embodiments, one immunoglobulin heterodimerization domain in each chain may be located amino-terminal to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain of each chain may be located carboxy-terminal to the immunoglobulin constant region of that chain. In other words, in these embodiments, the immunoglobulin constant region is located between the two immunoglobulin heterodimerization domains of each chain. As described herein, in certain embodiments, the CD3 binding polypeptides of the present invention comprise an immunoglobulin constant region (also referred to as a constant region) in the polypeptide chain. The incorporation of the immunoglobulin constant region delays the clearance of homodimeric and heterodimeric proteins formed by the two CD3 binding polypeptide chains from the circulation after administration to a subject. Through mutations or other alterations, the immunoglobulin constant region further allows for relatively easy modulation of the effector functions of the dimeric polypeptide (e.g., ADCC, ADCP, CDC, complement fixation, and Fc receptor binding), which can be either up or down depending on the disease, the treatment of which is carried out as is known in the art and described in this document. In certain embodiments, the immunoglobulin constant region of one or both of the polypeptide chains of the polypeptide homodimers and heterodimers of the present invention is capable of mediating one or more of these effector functions. In other embodiments, one or more of these effector functions are reduced or absent in the immunoglobulin constant region of one or both polypeptide chains of the polypeptide homodimers and heterodimers of the present invention compared to the corresponding wild-type immunoglobulin constant region. For example, in the case of dimeric CD3 binding polypeptides designed to stimulate PTCs, for example by incorporating a second binding domain, the immunoglobulin constant region may have reduced or no effector function compared to the corresponding wild-type immunoglobulin constant region.

The immunoglobulin constant region present in the CD3 binding polypeptides of the present invention may comprise or be derived from part or all of: a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof. For example, an immunoglobulin constant region may comprise a CH2 domain, a CH3 domain, CH2 and CH3 domains, CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains and a CH2 domain and part of a CH3- domain.

The CH2 domain that may form the immunoglobulin constant region of the CD3 binding polypeptide of the present invention may be a wild-type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain from certain immunoglobulin classes and subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD) and from various species (including human, mouse, rat and other mammals).

In certain embodiments, the CH2 domain is a wild-type human immunoglobulin CH2 domain, such as the wild-type human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD CH2 domains shown in SEQ ID NOs: 115, 199-201 and 195-197, respectively, in US 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the CH2 domain is the wild-type human IgG1 CH2 domain shown in SEQ ID NO: 115 of US 2013/0129723 (the sequence is incorporated herein by reference).

In certain embodiments, the CH2 domain is an altered immunoglobulin CH2 region (eg, an altered human IgG1 CH2 domain) that contains an amino acid substitution in asparagine at position 297 (eg, asparagine to alanine). This amino acid change reduces or abolishes glycosylation at this site and inhibits efficient Fc binding to FcyR and C1q. The sequence of the altered human IgG1 CH2 domain with the change from Asn to Ala at position 297 is shown in SEQ ID NO: 324 in US 2013/0129723 (the sequence is incorporated herein by reference). In certain embodiments, the CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human IgG1 CH2 domain) that contains at least one substitution or deletion at positions 234 to 238. For example, an immunoglobulin CH2 region may contain a substitution in positions 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238 , positions 234, 235, 237 and 238, positions 236-238, or any other combination of two, three, four or five amino acids at positions 234-238. In a further or alternative embodiment, the altered CH2 region may contain one or more (e.g., two, three, four, or five) amino acid deletions at positions 234-238, e.g., one of position 236 or position 237, while the other position is substituted. The above(s) mutation(s) reduce(s) or eliminate(s) the activity of antibody-dependent cell-mediated cytotoxicity (ADCC) or the ability to bind the Fc receptor of a polypeptide heterodimer that contains an altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues. In additional embodiments, only one of the amino acid residues at positions 234-238 has been deleted, while one or more of the remaining amino acids at positions 234-238 may have been replaced with another amino acid (eg, alanine or serine).

In certain other embodiments, the CH2 domain is an altered CH2 region of an immunoglobulin (e.g., an altered human IgG1 CH2 domain) that contains one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331. For example, the CH2 region the immunoglobulin may contain a substitution at positions 253, 310, 318, 320, 322 or 331, positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322 and 331. The above mutation(s) reduce(s) or eliminate(s) complement dependent cytotoxicity (CDC) of a polypeptide heterodimer that contains an altered CH2 domain.

In certain other embodiments, in addition to the amino acid substitution at position 297, the altered CH2 region (e.g., altered human IgG1 CH2 domain) may further comprise one or more (e.g., two, three, four, or five) additional substitutions at positions 234238. For example, The CH2 region of an immunoglobulin may contain a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any other combination of two, three, four or five amino acids at positions 234-238 in addition to position 297. In addition or alternatively the altered CH2 region may contain one or more (e.g., two, three, four, or five) amino acid deletions at positions 234-238, for example, at position 236 or position 237. Additional mutation(s) reduces(s) or eliminate(s) the activity of antibody-dependent cellular mediated cytotoxicity (ADCC) or the ability to bind the Fc receptor of a polypeptide heterodimer that contains an altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues. In additional embodiments, only one of the amino acid residues at positions 234-238 has been deleted, while one or more of the remaining amino acids at positions 234-238 may have been replaced with another amino acid (eg, alanine or serine).

In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region (e.g., an altered human IgG1 CH2 domain) in a fusion protein of the present invention may contain one or more than (eg, 2, 3, 4, 5, or 6) additional amino acid substitutions (eg, alanine substitutions) in

- 28 042409 one or more positions involved in complement fixation (for example, positions I253, H310,

E318, K320, K322 or P331). Examples of mutated immunoglobulin CH2 regions include human IgG1, IgG2, IgG4 and mouse IgG2a CH2 regions with substitutions for alanine at positions 234, 235,

237 (if available), 318, 320 and 322.

An example of a mutated immunoglobulin CH2 region is the murine IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320 and K322. In additional other embodiments, in addition to the amino acid substitution at position 297 and additional deletion(s) or substitution(s) at positions 234-238, the altered CH2 region (e.g., altered human IgG1 CH2 domain) may further comprise one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, an immunoglobulin CH2 region may contain (1) a substitution at position 297, (2) one or more substitutions or deletions, or a combination thereof, at positions 234-238 and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions I253, H310, E318, K320, K322, and P331, such as one, two, three replacements in positions E318, K320 and K322. The amino acids at the above positions may be substituted with alanine or serine. In certain embodiments, the immunoglobulin CH2 region comprises: (i) an amino acid substitution at position 297 of the asparagine and one amino acid substitution at position 234, 235, 236, or 237; (ii) an amino acid substitution at position 297 asparagine and amino acid substitutions at two of positions 234-237; (iii) an amino acid substitution at position 297 asparagine and amino acid substitutions at three of positions 234-237; (iv) amino acid substitution at position 297 asparagine, amino acid substitutions at positions 234, 235 and 237, and amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320, and 322.

Examples of altered immunoglobulin CH2 regions with amino acid substitutions at position 297 asparagine include: human IgG1 CH2 region with alanine changes at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO: 325 in US 2013/0129723, sequence indicated incorporated herein by reference), CH2 region of human IgG2 with alanine substitutions in V234, G236, and N297 (SEQ ID NO: 326 in US 2013/0129723, the sequence incorporated herein by reference), CH2 region of human IgG4 with alanine substitutions in F234, L235, G237, and N297 and a G236 deletion (SEQ ID NO: 322 in US 2013/0129723, the sequence incorporated herein by reference), human IgG4 CH2 region with alanine substitutions in F234, and N297 (SEQ ID NO: 343 in US 2013/0129723, the specified sequence is incorporated herein by reference), human IgG4 CH2 region with alanine substitutions in L235 and N297 (SEQ ID NO: 344 in US 2013/0129723, specified at sequence incorporated herein by reference), the human IgG4 CH2 region with alanine substitutions in G236 and N297 (SEQ ID NO: 345 in US 2013/0129723, said sequence incorporated herein by reference), and the human IgG4 CH2 region with substitutions for alanine in G237 and N297 (SEQ ID NO: 346 in US 2013/0129723, the specified sequence is incorporated herein by reference).

In certain embodiments, in addition to the amino acid substitutions described above, the altered CH2 region (e.g., altered human IgG1 CH2 domain) may contain one or more additional amino acid substitutions at one or more positions other than the above positions. Such amino acid substitutions may be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, P233 can be changed to E233 in an altered IgG2 CH2 region (see, for example, SEQ ID NO: 326 in US 2013/0129723, the sequence is incorporated herein by reference). Additionally or alternatively, in certain embodiments, the altered CH2 region may contain one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s), or substitution(s) may be located anywhere in the CH2 region of an immunoglobulin, for example, in the N or C terminus of the CH2 region of a wild-type immunoglobulin, resulting from the binding of a CH2 region to another region (eg, immunoglobulin heterodimerization domain binding domain) via a hinge region.

In certain embodiments, an altered CH2 region in a polypeptide of the present invention contains or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a CH2 region of a wild-type immunoglobulin, such as a CH2 region wild-type human IgG1, IgG2 or IgG4 or mouse IgG2a (eg IGHG2c).

The altered CH2 region of an immunoglobulin CD3 binding polypeptide of the present invention can be derived from the CH2 region of various immunoglobulin isotypes such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgD from various species (including human, mouse, rat and other mammals). In certain embodiments, the altered CH2 region of an immunoglobulin in a fusion protein of the present invention can be derived from the CH2 region of human IgG1, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c), the sequences of which are shown

- 29 042409 in SEQ ID NOs: 115, 199, 201 and 320 in US 2013/0129723 (these sequences are incorporated herein by reference).

In certain embodiments, the altered CH2 domain is a human IgG1 CH2 domain with substitutions for alanine at positions 235, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with substitutions L235A, E318A, K32OA, and K322A) (SEQ ID NO: 595 in US 2013/0129723, the specified sequence is incorporated herein by reference) and optionally the N297 mutation (eg, to alanine). In certain other embodiments, the altered CH2 domain is a human IgG1 CH2 domain with substitutions for alanine at positions 234, 235, 237, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with substitutions L234A, L235A, G237A , E318A, K32OA and K322A) (SEQ ID NO: 596 in US 2013/0129723, the specified sequence is incorporated herein by reference) and, optionally, the N297 mutation (eg, to alanine).

In certain other embodiments, the altered CH2 domain is an altered CH2 domain of human IgG1 with mutations known in the art to enhance immunological activity such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any their combination.

The CH3 domain that may form the immunoglobulin constant region of the CD3 binding polypeptide of the present invention may be a wild-type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain from certain immunoglobulin classes or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) of various species (including human, mouse, rat and other mammals). In certain embodiments, the CH3 domain is a wild-type human immunoglobulin CH3 domain, such as the wild-type human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH3 domains shown in SEQ ID NO: 116 , 208-210, 204-207, and 212, respectively, in US 2013/0129723 (these sequences are incorporated herein by reference). In certain embodiments, the CH3 domain is the wild-type human IgG1 CH3 domain shown in SEQ ID NO: 116 of US 2013/0129723 (the sequence is incorporated herein by reference). In certain embodiments, the CH3 domain is an altered CH3 domain of a human immunoglobulin, such as an altered CH3 domain based on or derived from the wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM antibodies. For example, the altered CH3 domain may be a human IgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions numbered according to EU numbering). Mutations at such positions may be involved in complement fixation. In certain other embodiments, the altered CH3 domain may be a human IgG1 CH3 domain, but with one or two amino acid substitutions at position F405 or Y407. Amino acids at these positions are involved in interaction with another CH3 domain. In certain embodiments, the altered CH3 domain may be an altered human IgG1 CH3 domain with the last lysine removed. The sequence of the altered CH3 domain is shown in SEQ ID NO: 761 in US 2013/0129723 (the sequence is incorporated herein by reference).

In certain embodiments, CD3-binding polypeptides forming a polypeptide heterodimer comprise a CH3 pair containing so-called ridge-to-trough mutations (see Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21, 1966). More specifically, mutations can be introduced into each of the two CH3 domains of each polypeptide chain such that the need for steric complementarity for CH3/CH3 binding causes the two CH3 domains to bind to each other. For example, a CH3 domain in a single chain polypeptide of a polypeptide heterodimer may contain the T366W mutation (an overhang mutation that replaces a small amino acid with a larger one), and a CH3 domain in another single chain polypeptide of a heterodimer polypeptide may contain a Y407A mutation (a trough mutation that replaces a large amino acid with a smaller one) . Other examples of lip-to-bottom mutations include (1) T366Y mutation in one CH3 domain and Y407T in another CH3 domain, and (2) T366W mutation in one CH3 domain and T366S, L368A, and Y407V mutations in another CH3 domain. .

The CH4 domain that can form the immunoglobulin constant region of the CD3 binding polypeptides of the present invention can be a wild-type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain from IgE or IgM molecules. In certain embodiments, the CH4 domain is a wild-type human immunoglobulin CH4 domain, such as the wild-type CH4 domains of human IgE or IgM molecules shown in SEQ ID NOs: 213 and 214, respectively, in US 2013/0129723 (these sequences are included in this document by reference). In certain embodiments, the CH4 domain is an altered CH4 domain of a human immunoglobulin, such as an altered CH4 domain based on or derived from the CH4 domain of human IgE or IgM molecules that have mutations that increase or decrease immunological activity associated with Fc- IgE or IgM region. In certain embodiments, the immunoglobulin constant region of the CD3 binding polypeptides of the present invention comprises a combination of CH2 domains,

- 30 042409

CH3 or CH4 (ie, more than one constant region domain selected from CH2, CH3, and CH4). For example, an immunoglobulin constant region may contain CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, an immunoglobulin constant region may contain two CH3 domains and no CH2 or CH4 domains (ie, only two or more CH3).

The multiple constant region domains that make up an immunoglobulin constant region may be based on or derived from the same immunoglobulin molecule or the same class or subclass of immunoglobulin molecules. In certain embodiments, the immunoglobulin constant region is IgG CH2CH3 (e.g., IgG1 CH2CH3, IgG2 CH2CH3, and IgG4 CH2CH3) and may be human (e.g., human IgG1, IgG2, and IgG4) CH2CH3. For example, in certain embodiments, an immunoglobulin constant region comprises (1) wild-type human IgG1 CH2 and CH3 domains, (2) human IgG1 CH2 with N297A (i.e., CH2(N297A)) and wild-type human IgG1 CH3 domains, or (3 ) CH2(N297A) of human IgG1 and altered CH3 of human IgG1 with the last lysine removed. Alternatively, the Multiple constant region domains may be based on or derived from different immunoglobulin molecules or different classes or subclasses of immunoglobulin molecules. For example, in certain embodiments, an immunoglobulin constant region contains both a human IgM CH3 domain and a human IgG1 CH3 domain. The multiple constant region domains that form the immunoglobulin constant region may be directly linked together, or may be linked to each other via one or more (eg, about 210) amino acids.

Examples of immunoglobulin constant regions are shown in SEQ ID NOs: 305-309, 321, 323, 341, 342, and 762 in US 2013/0129723 (these sequences are incorporated herein by reference).

In certain embodiments, the immunoglobulin constant regions of both CD3-binding polypeptides of a polypeptide homodimer or heterodimer are identical to each other. In certain other embodiments, the immunoglobulin constant region of one polypeptide chain of the heterodimeric protein is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, one immunoglobulin constant region of a heterodimeric protein may contain a node-mutated CH3 domain, while another immunoglobulin constant region of a heterodimeric protein may contain a trough-mutated CH3 domain.

This invention relates to CD3-binding proteins and polypeptides, which may contain any of the sequences shown in table. 14. Amino acid sequences for polypeptide constructs may or may not contain signal sequences. CD3 binding proteins may contain any of the CD3 binding domains described above. In some aspects, CD3 binding proteins comprise humanized V _H or V _L amino acid sequences, or both.

Examples of bispecific CD3-binding polypeptides are shown in table. 12 and 13. Such examples include anti-PSMA x anti-CD3 binding molecules (SEQ ID NOS: 62, 64, 66 and 68), anti-CD37 x anti-CD3 binding molecules (SEQ ID NOS: 72, 74, 76, 78, 80 and 82), anti-ROR1 x anti-CD3 binding molecules (SEQ ID NOS: 100, 104, 108, 112, 116 and 120) and anti-CD123 x anti-CD3 binding molecules (SEQ ID NOS: 197 and 198) .

CD3 binding molecules can be prepared using supports as generally described in US 2013/0129723 and US 2013/0095097, which are incorporated herein by reference in their entirety. CD3 binding proteins may contain two non-identical polypeptide chains, with each polypeptide chain containing an immunoglobulin heterodimerization domain. The docking domains of immunoglobulin heterodimerization are different. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In another embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is represented by Ck or Cλ isotypes or derivatives thereof.

The invention also includes nucleic acids (eg, DNA or RNA) encoding the CD3 binding domains, proteins or polypeptides described herein, or one or more polypeptide chains of the homodimeric or heterodimeric CD3 binding protein described herein. Nucleic acids according to the invention include nucleic acids containing a region that is essentially identical to the polynucleotide shown in table. 14 above. In certain embodiments, a nucleic acid according to the invention has at least about 80%, typically at least about 90%, and more often at least about 95% or at least about 98% identity with a polypeptide-encoding polynucleotide, given in table. 14. Nucleic acids of the invention also include complementary nucleic acids. In some cases, sequences become fully complementary (no mismatches) when aligned. In other cases, the sequences may have up to about 20% mismatch. In some embodiments, nucleic acids are provided that encode both the first and second

- 31 042409 heterodimeric CD3-binding protein chain according to the invention. Nucleic acids provided herein can be generated using codon optimization, sequence degeneracy, silent mutations, and other DNA techniques to optimize expression in a particular host, and the present invention includes such sequence modifications.

The present invention relates to an isolated nucleic acid molecule encoding the CD3 binding domains, proteins and polypeptides (or parts thereof) described herein, said nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59. Polynucleotide molecules containing the desired polynucleotide sequence are propagated by placing the molecule in a vector. Use of viral and non-viral vectors, including plasmids. The choice of plasmid depends on the type of cell in which propagation is desired and the purpose of propagation. Some vectors are used to amplify and obtain large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Other vectors are suitable for transfer and expression in cells in an animal or human body. The choice of an appropriate vector is within the skill of the art. Many such vectors are commercially available. The partial or full length polynucleotide is inserted into the vector, typically by attaching a DNA ligase to a restriction enzyme cleavage site in the vector. Alternatively, the desired nucleotide sequence can be inserted by in vivo homologous recombination. Typically, this is done by attaching regions of homology to the vector on the sides of the required nucleotide sequence. Regions of homology are added, for example, by ligation of oligonucleotides or by polymerase chain reaction using primers containing both the region of homology and part of the desired nucleotide sequence.

An expression cassette or system can be used for expression. In order to express a nucleic acid encoding a polypeptide as described herein, a polypeptide encoding nucleic acid molecule operably linked to regulatory sequences that regulate transcriptional expression in an expression vector is introduced into a host cell. In addition to transcriptional control sequences such as promoters and enhancers, expression vectors may contain translational control sequences and a marker gene that is useful for selecting cells carrying the expression vector. The gene product encoded by a polynucleotide of the invention is expressed in any convenient expression system, including, for example, those of bacteria, yeast, insects, amphibians, and mammals. In the expression vector, the polynucleotide encoding the polypeptide is suitably linked to a regulatory sequence to obtain the desired expression properties. These sequences include promoters, enhancers, terminators, operators, repressors, and inducers. Promoters can be regulatable (eg, the promoter from the steroid-inducible pIND vector (Invitrogen)) or constitutive (eg, promoters from CMV, SV40, elongation factor, or PrEP sequences). They are linked to the desired nucleotide sequence using the vector binding methods described above. Any methods known in the art may be used. Accordingly, in general, an expression vector will provide a transcription and translation initiation region, which may be inducible or constitutive, with the coding region operably linked under transcriptional control by the transcription initiation region and the transcription and translation termination region.

The expression cassette (expression unit) can be introduced into a variety of vectors, e.g., plasmid, BAC, YAC, bacteriophage such as lambda, P1, M13, etc., plant or animal viral vectors (e.g., retroviral vectors, adenovirus vectors) and the like, where the vectors are generally characterized by the ability to select cells containing the expression vectors. Vectors can provide extrachromosomal support, particularly in the case of plasmids or viruses, or integration into the host's chromosome. If extrachromosomal support is needed, a starting sequence for plasmid replication is provided, which may be low or high copy number. A wide range of markers are available for screening, in particular those that protect against toxins, more specifically antibiotics. The particular marker chosen is chosen according to the nature of the host organism, and in some cases complementation with auxotrophic hosts may be used. When introducing the DNA construct, any convenient method may be used, including, for example, conjugation, bacterial transformation, DNA calcium precipitation, electroporation, fusion, transfection, infection with viral vectors, biolistics, and the like. This invention relates to an expression vector containing a nucleic acid segment, and the specified nucleic acid segment may contain the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 59.

Accordingly, proteins for use in the present invention can be produced in genetically engineered host cells in accordance with conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured cells of higher eukaryotes (including cultured cells of multicellular organisms), in particular cultured mammalian cells. . Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001) and Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999). For example, for recombinant expression of a homodimeric CD3 binding protein containing two identical CD3 binding polypeptides described herein, the expression vector will generally contain a nucleic acid segment encoding a CD3 binding polypeptide operably linked to a promoter. For recombinant expression of a heterodimeric CD3 binding protein containing different first and second polypeptide chains, the first and second polypeptide chains can be co-expressed from two separate vectors in a host cell to express the entire heterodimeric protein. Alternatively, for the expression of heterodimeric CD3 binding proteins, the first and second polypeptide chains are co-expressed from different expression units in the same vector in the host cell to express the entire heterodimeric protein. The expression vector(s) are introduced into the host cell by conventional methods, and then the transfected cells are cultured by conventional methods to obtain the encoded polypeptide(s) to obtain the corresponding CD3-binding protein.

To direct the recombinant protein into the secretory pathway of the host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. The secretory signal sequence may be the sequence of the native form of the recombinant protein or may be derived from another secreted protein or synthesized de novo. The secretory signal sequence is operably linked to the polypeptide-encoding DNA sequence, i.e. the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized protein into the secretory pathway of the host cell. Secretory signal sequences are usually located 5' to the DNA sequence encoding the polypeptide of interest, although some signal sequences may be located elsewhere in the DNA sequence of interest (see, for example, Welch et al., US patent No. 5037743; Holland et al., US patent No. 5143830). In certain variations, the secretory signal sequence for use in accordance with the present invention has the amino acid sequence MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 195).

Cultured mammalian cells are suitable hosts for the production of recombinant proteins for use in the present invention.

Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456 , 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15 :73, 1993; Ciccarone et al., Focus 15:80, 1993). Obtaining recombinant polypeptides in cultured mammalian cells is described, for example, in Levinson et al., US patent No. 4713339; Hagen et al., US patent No. 4784950; Palmiter et al., US patent No. 4579821; and Ringold, US Pat. No. 4,656,134. Examples of suitable mammalian host cells include African green monkey kidney (Vero; ATCC CRL 1587), human primary kidney (293-HEK; ATCC CRL 1573), newborn hamster kidney (BHK-21 , BHK-570; ATCC CRL 8544, ATCC CRL 10314), dog kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (Cho-Κι; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitocytes (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H -4-P-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658). Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, Virginia. You can use strong transcriptional promoters, such as promoters from SV-40 or cytomegalovirus. See, for example, US Pat. No. 4,956,288. Other suitable promoters include those from the metallothionein genes (US Pat. Nos. 4,579,821 and 4,601,978) and the adenoviral major late promoter.

Drug susceptibility selection is generally used to select cultured mammalian cells that have been introduced with foreign DNA. Such cells are commonly referred to as transfectants. Cells which are cultured in the presence of a selective agent and which are able to pass the gene of interest to their progeny are called stable transfectants. Examples of selectable markers include a gene encoding resistance to the antibiotic neomycin, which allows selection in the presence of a neomycin drug.

- 33 042409 of the first preparation, such as G-418 or the like; the gpt gene for xanthine-guanine phosphoribosyltransferase, which allows host cells to grow in the presence of mycophenolic acid/xanthine; and markers that confer resistance to zeocin, bleomycin, blastocidin and hygromycin (see, for example, Gatignol et al., Mol. Gen. Genet. 207:342, 1987; Drocourt et al., Nucl. Acids Res. 18:4009 , 1990). Breeding systems can also be used to increase the level of expression of a gene of interest, which is called amplification. Amplification is carried out by culturing the transfectants in the presence of a low level of selective agent and then increasing the amount of selective agent to select cells that produce high levels of the products of the introduced genes. An example of an amplifiable selectable marker is dihydrofolate reductase, which provides resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells, and avian cells. The use of Agrobacterium rhizogenes as a vector for gene expression in plant cells has been described in Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides in them are described in Guarino et al., US 5162222 and WO 94/06463.

Insect cells can be infected with a recombinant baculovirus, usually derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). See King and Possee, The Baculovirus Expression System: A Laboratory Guide (Chapman & Hall, London); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press., New York 1994); and Baculovirus Expression Protocols. Methods in Molecular Biology (Richardson ed., Humana Press, Totowa, NJ, 1995). Recombinant baculovirus can also be generated using the transposon based system described in Luckow et al. (J. Virol. 67:4566-4579, 1993). This system, which uses transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, MD). The transfer vector (eg, PFASTBAC1; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into the baculovirus genome found in E. coli as a large plasmid called a bacmid. See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfer vectors may include an in-frame fusion to DNA encoding a polypeptide extension or affinity tag as described above. Using techniques known in the art, a transfer vector containing a protein-coding DNA sequence is transformed into E. coli host cells and the cells are screened for bacmids that contain the discontinuous lacZ gene indicative of the presence of recombinant baculovirus. Bacmida DNA containing the recombinant baculovirus genome is isolated using conventional techniques and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. A recombinant virus is then generated that expresses the protein of interest. Stocks of recombinant CD3 viruses are generated by methods commonly used in the art.

In the case of protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the Fall Armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVE cells; Invitrogen, Carlsbad, CA ). In general, see Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994). See also US Pat. No. 5,300,435. Serum-free media are used to grow and maintain cells. Suitable media formulations are known in the art and can be purchased from commercial suppliers. Cells are grown from an inoculation density of approximately 2-5x105 cells to a density of 1-2x106 cells at which time a stock of recombinant virus is added at a MOI of 0.1 to 10, more typically approximately 3. The procedures used are generally described in available laboratory manuals (see, for example, King and Possee, supra; O'Reilly et al., supra; Richardson, supra).

Fungal cells, including yeast cells, can also be used within the scope of the present invention. In this regard, yeast cells include, for example, Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and obtaining recombinant polypeptides from them are described, for example, in Kawasaki, US patent No. 4599311; Kawasaki et al., US Pat. No. 4,931,373; Brake, US Pat. No. 4,870,008; Welch et al., US Pat. No. 5,037,743; and Murray et al., US Pat. No. 4,845,075. Transformed cells are selected for phenotype as determined by a selectable marker, typically drug resistance or ability to grow in the absence of a particular nutrient (eg, leucine). An example of a vector system for use in Saccharomyces cerevisiae is the POT1 vector system described in Kawasaki et al. (US patent No. 4931373), which allows the selection of transformed cells by growing in a medium containing glucose. Suitable promoters and terminators for use in yeast include those derived from glycolytic enzyme genes (see, for example, Kawasaki, patent

- 34 042409

US No. 4599311; Kingsman et al., US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also US Pat. Nos. 4,990,446; 5063154; 5139936; and 4661454.

Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica,

Pichia guillermondii and Candida maltosa are known in the art.

See, for example, Gleeson et al., J. Gen. microbiol. 132:3459-3465, 1986; Cregg, US patent No. 4882279; and Raymond et al., Yeast 14:11-23, 1998. Aspergillus cells can be used according to the methods of McKnight et al., US Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are described in Sumino et al., US Pat. No. 5,162,228. Methods for transforming Neurospora are described in Lambowitz, US Pat. No. 4,486,533. The preparation of recombinant proteins in Pichia methanolica is described in US Pat. Nos. 5,716,808; 5736383; 5854039; and 5888768.

Prokaryotic host cells, including bacterial strains of Eschehchia coli, Bacillus, and other genera, are also suitable host cells for the purposes of the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned into them are well known in the art (see, for example, Sambrook and Russell, supra). When the recombinant protein is expressed in bacteria such as E. coli, the protein may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by the bacterial secretion sequence. In the first case, the cells are lysed and the beads are reduced and denatured using, for example, guanidine isothiocyanate or urea. The denatured protein can then be refolded and dimerized by dilution of the denaturant, for example by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against saline buffer. Alternatively, the protein can be recovered from the cytoplasm in a soluble form and isolated without the use of denaturants. The protein is recovered from the cell as an aqueous extract, for example, in phosphate-buffered saline. To capture the protein of interest, the extract is applied directly to a chromatographic medium such as an immobilized antibody or heparin Sepharose column. Secreted proteins can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (through, for example, sonication or application of osmotic shock) to release the contents of the periplasmic space and restore the protein, thereby eliminating the need for denaturation and refolding. Antibodies, including single chain antibodies, can be produced in bacterial host cells according to known methods. See, for example, Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. sci. USA 85:5879-5883, 1988; and Pantoliano et al., Biochem. 30:10117-10125, 1991.

The transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components necessary for the growth of the selected host cells. A variety of suitable media are known in the art, including certain media and complex media, which typically contain a carbon source, a nitrogen source, essential amino acids, vitamins, and minerals. Media may also contain components such as growth factors or serum, if necessary. In general, the growth medium provides for the selection of cells containing exogenously added DNA, for example, by selection for drug susceptibility or the creation of an essential nutrient deficiency, which is complemented by a selectable marker that is carried by an expression vector, or that is co-transfected into the host cell. . CD3 binding proteins can be purified by conventional protein purification methods, typically through a combination of chromatographic methods. In general, see Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer-Verlag, New York 1994). Proteins containing the immunoglobulin Fc region can be purified by affinity chromatography on immobilized protein A or protein G. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purification or to provide desalting, buffer exchange, and the like.

The CD3 binding molecules described herein can be used in a method for treating a subject (eg, a human or non-human primate) or for the manufacture of a medicament for treating a subject. In general, such methods include administering to a subject in need of such treatment the CD3 binding protein described herein. The CD3 binding molecules described herein can be used in a method for treating a subject (eg, a human or non-human primate) or for the manufacture of a medicament for treating a subject. In general, such methods include administering to a subject in need of such treatment the CD3 binding protein described herein. In some embodiments, the CD3 binding protein has at least one effector function selected from antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CCC), such that the CD3-binding protein induces ADCC and/or CCC against CD3-expressing cells in organism - 35 042409 me of the subject. In some aspects, the present invention provides methods for treating a subject with a disorder characterized by CD3 overexpression. In one instance, a monospecific CD3 binding polypeptide is administered to a patient suffering from an autoimmune disease (eg, rheumatoid arthritis). In certain variations, the CD3 binding protein provided herein can be used to modulate T cell function and behavior, thus providing a therapeutic treatment for T cell mediated disease, including autoimmune or inflammatory diseases in which T cells play a significant role. Because the CD3 binding proteins of the present invention do not activate T cells and/or induce cytokine release, they have an advantage over other molecules directed against the TCR complex (e.g., anti-CD3 antibodies) in that they do not have the characteristic or characterized by reduced side effects such as cytokine release syndrome and acute toxicity. In another instance, the CD3 binding polypeptide is administered to a subject prior to organ transplantation.

In another aspect, the present invention provides a method of treating a disorder characterized by overexpression of a tumor antigen, such as cancer. Examples of tumor antigens that can be recognized by bispecific CD3 binding proteins include PSMA, CD19, CD20, CD37, CD38, CD123, Her2, ROR1, RON, A33 glycoprotein antigen (gpA33), and CEA. In general, such methods include administering to a subject in need of such treatment a therapeutically effective amount of a CD3 binding protein containing a second binding domain that binds a tumor antigen, as described herein. In some embodiments, the CD3 binding protein induces redirected T-cell cytotoxicity (PTCC) against tumor antigen-expressing cells in a subject. Examples of cancers that are treatable according to the present invention include, for example, prostate cancer, colorectal cancer, renal cell carcinoma, bladder cancer, salivary gland cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, melanoma, breast cancer. (eg, triple-negative breast cancer), adrenal cancer, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute myelocytic leukemia (AML), B-lymphoid leukemia, plasmacytoid dendritic cell neoplasm (PDC), and leukemia hairy cells.

The invention also provides methods for treating cancer or an autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of the compositions or CD3 binding polypeptides described herein.

In some embodiments, the invention provides a method of treating a patient with cancer comprising administering to the patient a CD3 binding polypeptide comprising a CD3 binding domain that specifically binds to human CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is (a) at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 88; or (b) at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 89; and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO: 86. In some embodiments, for the implementation of the methods of treatment and uses described herein, the CD3 binding protein is delivered in a manner consistent with conventional techniques associated with the management of the disease or disorder being treated. In accordance with the invention described herein, a therapeutically effective amount of a CD3 binding protein is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

Subjects for administration of the CD3 binding proteins described herein include patients at high risk of developing a particular disorder, as well as patients who have such a disorder. Typically, the subject has been diagnosed with a disorder for which treatment is envisaged. In addition, during the course of treatment, subjects can be monitored for any change in the disorder (eg, an increase or decrease in the clinical symptoms of the disorder). Also, in some variations, the subject does not suffer from another disorder,

- 36 042409 buoyant treatment that includes targeting CD3-expressing cells.

In prophylactic use, pharmaceutical compositions or medicaments are administered to a patient who is susceptible or otherwise at risk for a particular disorder in an amount sufficient to eliminate or reduce the risk or delay the onset of the disorder. In therapeutic use, the compositions or medicaments are administered to a patient suspected of or already suffering from such a disorder in an amount sufficient to treat, or at least partially suppress, the symptoms of the disorder and its complications. The amount required for this is referred to herein as a therapeutically effective dose or amount. In both prophylactic and therapeutic regimens, agents are typically administered at multiple dosages until a sufficient response is achieved. As a rule, this response is monitored and repeated doses are used when the desired response is attenuated.

To identify patients for treatment in accordance with the methods of the invention, conventional research methods can be used to determine the risk factors associated with specific disorders, or to determine the status of an existing disorder identified in a subject. Such methods may include, for example, determining whether the individual has relatives who have been diagnosed with a particular disorder. Research methods may also include, for example, conventional research to determine marital status for a particular disorder that has a hereditary component. For example, it is also known that different types of cancer have certain non-inherited components. Non-inherited components of cancer include, for example, mutations in a number of genes that are transformative (eg, Ras, Raf, EGFR, cMet, and others), the presence or absence of certain HLA molecules and cytotoxicity suppression receptors (KIRs), or the mechanisms by which cancer cells able to modulate the immune suppression of cells like NK cells and T cells, both directly and indirectly (see, e.g., Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp Immunol 149:1-8, 2007). To this end, nucleotide probes can usually be used to identify individuals carrying genetic markers associated with a particular disorder of interest. In addition, a wide range of immunological methods are known in the art for detecting markers of a particular disorder. For example, various ELISA immunoassay methods are available and well known in the art that use monoclonal antibody probes to detect antigens associated with specific tumors. screening can be performed as indicated with known symptoms, age factors, associated risk factors for the patient, etc. These methods allow the attending physician to easily select for treatment patients in need of the methods described herein. According to these methods, targeting of abnormal tumor antigen-expressing cells can be performed as an independent treatment program or as a concomitant, adjunct or coordinated treatment regimen along with other treatment options.

For administration, the CD3 binding protein can be formulated into a pharmaceutical composition. The pharmaceutical composition may contain: (i) a CD3 binding polypeptide; and (ii) a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition containing a CD3 binding protein can be formulated according to known methods for the preparation of pharmaceutically applicable compositions wherein the therapeutic molecule is in admixture with a pharmaceutically acceptable carrier, diluent or excipient. A carrier is said to be a pharmaceutically acceptable carrier if its administration is tolerable to the patient receiving it. One example of a pharmaceutically acceptable carrier is sterile phosphate buffered saline. Other suitable carriers, diluents or excipients are well known to those skilled in the art. (See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) The formulations may additionally include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on surfaces vials, etc.

The pharmaceutical composition may be formulated in a dosage form selected from the group consisting of: oral unit dosage form, intravenous unit dosage form, intranasal unit dosage form, suppository unit dosage form, intradermal unit dosage form, intramuscular unit dosage form, intraperitoneal unit dosage form dosage form, subcutaneous dosage unit form, epidural dosage unit form, sublingual dosage unit form, and intracerebral dosage unit form. The oral unit dosage form may be selected from the group consisting of: tablets, pills, wafers, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols and sprays.

A pharmaceutical composition containing a CD3 binding protein therapeutic may be administered to a subject in a therapeutically effective amount. According to spo

- 37 042409 Thus according to the present invention, the CD3-binding protein can be administered to subjects via various routes of administration, including, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, parenteral, intranasal, intra-pulmonary, transdermal, interpleural, intrathecal and oral routes of administration. For prevention and treatment purposes, the antagonist can be administered to the subject as a single bolus delivery, by continuous delivery (e.g., continuous transdermal delivery) over an extended period of time, or according to a repeated administration protocol (e.g., on an hourly, daily, weekly, or monthly basis).

In this context, the determination of effective dosages is generally based on animal model studies followed by human clinical studies and is guided by the determination of effective dosages and administration protocols that substantially reduce the occurrence or severity of subject disease in model subjects. Effective dosages of the compositions of the present invention will vary depending on a variety of different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other medications administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself, and its ability to evoke the desired response in the individual. Typically, the patient is a human, but in the case of certain diseases, the patient may be a non-human mammal. Typically, dosing regimens are adjusted to provide the optimal therapeutic response, i.e. optimize safety and efficiency. Accordingly, a therapeutically effective amount is also one in which any undesirable side effects are outweighed by the beneficial effect of administration of the CD3 binding protein described herein. For administration of the CD3 binding protein, the dosage may range from about 0.1 µg to 100 mg/kg, or from 1 µg/kg to about 50 mg/kg, and more typically from 10 µg to 5 mg/kg of the subject's body weight. In more specific embodiments, the effective amount of the agent is from about 1 µg/kg to about 20 mg/kg, from about 10 µg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 5 mg/kg. Dosages within this range may be achieved by one or more administrations, including, for example, a number of administrations per day or daily, weekly, biweekly or monthly administration. For example, in certain variations, the regimen consists of an initial administration followed by a number of consecutive administrations at weekly or biweekly intervals. Another regimen consists of an initial administration followed by a number of consecutive administrations at monthly or bi-monthly intervals. Alternatively, administration may be on an irregular basis as indicated based on observed clinical symptoms of the disorders.

The dosage of the pharmaceutical composition may be varied by the attending physician to maintain the desired concentration at the target site. For example, when choosing an intravenous route of administration, the local concentration of the agent in the bloodstream in the target tissue can be approximately 0.01-50 nanomoles of the composition per liter, sometimes from about 1.0 nanomoles per liter to 10, 15 or 25 nanomoles per liter, depending on the status of the subject and the intended measured response. Higher or lower concentrations can be selected based on the method of delivery, for example, transepidermal delivery and delivery through the mucosal surface. Dosage should also be adjusted based on the release rate of the formulation being administered, e.g. nasal spray and powder, sustained release oral or injectable particles, transdermal formulations, etc. To achieve the same level of serum concentration, e.g. slow release particles with a release rate of 5 nanomoles (under standard conditions) would have to be administered at a dosage approximately twice that of particles with a release rate of 10 nanomoles.

The Ahtu-CD3 therapeutic agent (eg, CD3 binding protein) may also be administered at a daily dosage of about 0.001 to about 10 milligrams (mg) per kilogram (mg/kg) of body weight, preferably administered as a single daily dose or divided doses two to six times a day. When administered to an adult human patient, a therapeutically effective amount may be administered at doses ranging from 0.2 mg to 800 mg per dose, including, but not limited to, 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose. dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and multiple, usually consecutive daily doses may be administered during the course of treatment. Ahtu-CD3 therapeutic agent can be administered at different times of the day. In one embodiment, the optimal therapeutic dose may be administered in the evening. In another embodiment, the optimum therapeutic dose may be administered in the morning. The total daily dosage of the anti-CD3 therapeutic agent may, in one embodiment, be in the range of about 1 mg to about 2 g, and is often in the range of about 100 mg to about 1.5 g, and most often is in the range of about 200 mg. up to about 1200 mg. In the case of a typical 70 kg adult human, the total daily dose of an anti-CD3 therapeutic agent can range from about 2 mg to about 1200 mg, and is often in the range, as above, from about 0.2 mg to about 800 mg. mg.

With regard specifically to the treatment of solid tumors, protocols for evaluating endpoints and antitumor activity are well known in the art. Although tumor response evaluation may be defined differently in each protocol, the RECIST criteria (Response evaluation Criteria in solid tumors) are currently considered the recommended guidelines for tumor response evaluation by the National Cancer Institute (see Therasse). et al., J Natl Cancer Inst 92:205-216, 2000). According to the RECIST criteria, tumor response means the reduction or elimination of all detectable lesions or metastases. In general, a disease is considered detectable if it includes lesions for which accurate measurements can be made in at least one dimension with a result > 20 mm in the case of conventional methods or > 10 mm in the case of helical CT scanning, with edges clearly defined by the method. medical photography or radiography, computed axial tomography (CT), magnetic resonance imaging (MRI) or clinical examination (in case of external lesions). Undetectable disease means that the disease consists of lesions < 20 mm in the case of traditional methods or < 10 mm in the case of helical CT scanning and truly unmeasurable lesions (too small for accurate measurement). Undetectable disease includes pleural effusions, peritoneal effusions, and disease supported by indirect evidence.

Objective status criteria are required for protocols to evaluate the response of solid tumors. Typical criteria include the following: (1) complete response (CR), defined as the complete disappearance of the disease being detected; no new lesions; absence of disease-related symptoms; no evidence of undetectable disease; (2) partial response (PR), defined as a 30% total reduction in the largest diameter of the target lesions; (3) progressive disease (PD), defined as a 20% cumulative increase in the largest diameter of the target lesions or the appearance of any new lesions; (4) stable disease or no response, defined as not falling under the definition of PO, OR, or progressive disease. (See Therasse et al., supra.) Additional endpoints accepted in the field of oncology engineering include overall survival (OS), disease-free survival (FSDS), objective response rate (ORR), time to progression (TRT), and progression-free survival (PFS) (see Guidance for Industry: Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies, April 2005, Center for Drug Evaluation and Research, FDA, Rockville, MD.)

Pharmaceutical compositions can be supplied as a kit containing a container that contains the pharmaceutical composition described herein. The pharmaceutical composition may be, for example, in the form of an injectable solution for one or more doses or as a sterile powder, which is reconstituted before injection. Alternatively, such a kit may include a dry powder nebulizer, a liquid aerosol generator, or a nebulizer for administering the pharmaceutical composition. Such a kit may further contain written information about the indications and use of the pharmaceutical composition.

The present invention will be better understood with the aid of the following examples, which are purely illustrative and in no way limit the invention.

Examples

Example 1 Construction of Stabilized CD3 Binding Molecules.

To improve the thermal stability of the CD3 binding molecule DRA222, an engineered version of the humanized Cris7 antibody, the variable domains of Cris7 were rehumanized using alternative human germline framework sequences. The heavy chain variable domain of DRA222 corresponds to SEQ ID NO: 87 and the light chain variable domain of DRA222 corresponds to SEQ ID NO: 90. Fc DRA222 is sometimes referred to as TSC311 or TSC312 (amino acid sequence according to SEQ ID NO: 2; nucleotide sequence according to SEQ ID NO: 1 ). See, Reinherz E.L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986) regarding the description of the parent antibody Cris7. Additional changes have also been made to improve affinity and thermal stability.

Ways.

The following methods were used to obtain the results shown in this example.

Differential Scanning Calorimetry (DSC).

Thermograms for recombinant proteins purified by standard purification methods were obtained on a GE VP-Capillary DSC equipped with an autosampler. Approximately 550 μl of each sample (typically 0.5 mg/ml) in PBS was injected into the sample in a capillary manner using PBS as a control in the second capillary. The analysis was carried out at temperatures from 25°C to 130°C with a heating rate of 1°C per minute. The feedback was set to low and the sampling time used was 8 ms. Data analysis was performed using Origin. The sample thermogram was corrected for buffer heat capacity by subtracting the previous buffer/buffer scan using a formulation stock solution and normalized based on sample concentration and corrected for background.

- 39 042409

Differential scanning fluorimetry (DSF).

Thermograms for recombinant proteins purified by standard purification methods were also obtained in a high-throughput format by performing DSF analysis on a real-time PCR device (Bio-Rad iCycler iQ5). Approximately 40 µl of each sample at 0.8 mg/ml in PBS was mixed with 5 µl of pre-diluted SYPRO orange dye (Catalog # S-6650, Life Technologies). The established protocol for obtaining the melting curve corresponded to a temperature increase from 25 to 90°C, in steps of 0.2°C. Fluorescent signals were recorded using a Texas Red fluorescent dye filter set, which consists of a 575/30X excitation filter and a 620/30M emission filter. The resulting fluorescence intensity data was exported to Prism 6 data analysis software (GraphPad Software, Inc.). The thermal Tm value for the protein was calculated as the temperature when the second derivative of the fluorescence intensity with respect to temperature is −d(RUF)/dT2=0.

Flow cytometry for Jurkat human T cells.

Binding studies were performed using standard flow cytometric staining procedures using the CD3 ⁺ Jurkat T cell line. All labeling and washing procedures were performed in 96-well U-bottom plates in saline buffer with 3% BSA and 2 mM EDTA. Jurkat cells were seeded at a density of 200,000 cells per well and incubated with a range of concentrations of the studied molecules from 0.1 to 200 nM in 50 μl volume/well for 30 min on ice. Cells were washed three times and then incubated for an additional 30 min on ice with fluorescently labeled minimally cross-reactive secondary polyclonal antibody, F(ab') ² goat anti-human IgG, (Jackson ImmunoResearch Laboratories) and 7-AAD viability dye. . The cells were then washed twice and the samples analyzed on a BD LSRII flow cytometer. Sample files were analyzed using FlowJo software; the average fluorescence intensity (CIF) of the live population of Jurkat cells in each well was calculated after gating on live cells (forward and side scatter, then 7-AAD ^- cells).

Modeling of homology, analysis of susceptibility to spatial aggregation.

In the Accelrys Discovery Studio 4.0 program, homology models for variable domains were constructed using the Annotate Antibody Sequence (annotate the antibody sequence), Identify Framework Templates (identify framework matrices), Model Antibody Framework (model an antibody framework) and Model Antibody Loops (model antibody loops) protocols. . Spatial aggregation susceptibility analysis was also performed in Accelrys Discovery Studio 4.0 using the Calculate Aggregation Scores protocol.

Chromium release assay for human T cells.

Target positive tumor cell lines (MDA-MB-231, Kasumi-2, C4-2B and Ramos cell lines) were cultured according to the protocols provided. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a standard ficoll gradient. The isolated cells were washed in saline buffer. T cells were further isolated using the Pan T-cell Isolation Kit (catalog #130-096-535, Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's protocol. The isolated T cells were aliquoted and stored in liquid nitrogen for a long time. Pre-prepared T cells were thawed the day before analysis in warm RPMI medium with 10% human serum. During the assay, concentrations of bispecific molecules were added to pre-prepared T cells (approximately 100,000 per well) with a final concentration ranging from 200 to 0.01 pM. Total lysis control was provided by including 0.04% NP-40 as a treatment. Approximately 2.5x106 target cells were treated with 0.125 mCi ⁵¹ Cr and incubated for 90 min at 37° C., in a humidified incubator with 5% CO ₂ . After incubation, the cells were washed 4 times with diluted assay medium (RPMI with 1% human serum) and resuspended in 12.5 ml of complete assay medium (RPMI with 10% human serum). From this suspension, 50 μl per well was dispensed into 96-well U-bottomed plates (approximately 10,000 cells per well) to give a total volume of 200 ml per well and a ratio between T cells and target cells of 10:1. Zero lysis control was provided only by target cells, excluding T cells.

The plates were incubated for 4 h (and occasionally also for 24 h) at 37°C, 5% CO ₂ in a humidified incubator, after which they were centrifuged at 1000 rpm for 3 min and 25 μl of the supernatant was transferred from each well. into the appropriate well of a Luma 96-well sample plate. The samples were allowed to air dry in a chemistry cabinet for 18 h and then the radioactivity was counted with a TopCount microplate scintillation counter (PerkinElmer) using a standard protocol.

The percentage of specific lysis was calculated using the formula:

((signal in drug-treated sample - background signal from samples containing only target cells) / (signal in wells for assessing total lysis - background signal from samples containing only target cells)) x 100.

- 40 042409

Flow cytometry for cynomolgus macaque T cells.

Cynomolgus monkey peripheral blood collected in heparin tubes was transported overnight from a supplier (Charles River laboratories). After preparation, peripheral blood cells (PBMCs) were isolated using density gradient separation tubes (CPT tubes, Beckton Dickinson). Blood was diluted 1:1.5 in saline buffer before being transferred to CPT tubes. The CPT tubes were centrifuged and the separated PBMC population was collected and washed with saline buffer containing 0.2% BSA and 5 nM EDTA. The remaining red blood cells in the preparation were lysed using a lysis buffer based on ammonium chloride and potassium for red blood cells. The cells were washed for additional time to remove the remaining platelets. The labeling and washing steps of PBMCs were performed in 96-well U-bottom plates in saline buffer with 0.2% BSA and 2 mM EDTA. PBMCs were seeded at a density of 200,000 cells per well and incubated with a range of concentrations of the studied molecules from 0.1 to 300 nM in 50 μl volume/well for 30 min on ice. Cells were washed three times and then incubated for an additional 30 min on ice with fluorescently labeled antibodies to CD2 and CD16 of non-human primates (Biolegend), anti-idiotypic antibodies to anti-PSMA or anti-CD37 binding domains, and 7-AAD viability dye. . Samples were washed twice, fixed for 20 minutes on ice with 1% formaldehyde in saline, washed again and analyzed on a BD LSRII flow cytometer. Sample files were analyzed using FlowJo software; the average fluorescence intensity (FIR) of the binding of the studied molecules on T cells in each well was calculated after gating on live T cells (forward and side scatter, 7-AAD ^- , CD2 ⁺ CD16 ^- cells).

Chromium release analysis for cynomolgus monkey PBMC.

C4-2B and Ramos cell lines were cultured according to the protocols provided. Peripheral blood mononuclear cells (PBMCs) were isolated from cynomolgus monkey peripheral blood using BD VACUTAINER® CPT™ blood product tubes with sodium heparin (Cat. #362753). The isolated cells were washed in saline buffer. Concentrations of bispecific molecules were added to isolated PBMCs (approximately 100,000) with a final concentration ranging from 10,000 to 0.0128 pM. Total lysis control was provided by including 0.04% NP-40 as a treatment. Approximately 5x106 target C4-2B cells or Ramos cells were treated with 0.25 mCi ⁵¹ Cr and incubated for 75 min at 37°C. After incubation, cells were washed 4 times with cell culture medium (RPMI with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, Na glutamine and 20 mM HEPES) and resuspended in 25 ml of medium. From this suspension, 50 μl per well was dispensed into 96-well U-bottomed plates (approximately 10,000 cells/well) to obtain a ratio between T cells and target cells of 10:1. The plates were incubated for 4 hours at 37°C, 5% CO ₂ in a humidified incubator, after which they were centrifuged at 225 rpm for 3 minutes and 25 μl of the supernatant was transferred from each well to the appropriate well of a 96-well sample plate Luma. The samples were allowed to air dry in a chemical cabinet for 18 h and then the radioactivity was counted with a TopCount scintillation counter using a standard protocol.

Results.

Stage 1.

CD3 generation of original humanized CD3 binding constructs.

Cris7 variable domains were rehumanized using four human germline variable heavy sequences (IGHV1-2*02 (H7), IGHV1-46*02 (H8), IGHV13*01(H9) and IGHV1-69*02 (H10)) and 2 human germline variable light sequences (IGKV3-11*01 (L4) and IGKV1-33*01 (L5)), based on sequence homology. A total of 12 single chain variable fragment (scFv) constructs were generated in antiCD3 scFv Fc format using the H75 linker (QRHNNSSLNTGTQMAGHSPNS; SEQ ID NO: 148) (Table 3). The sequences of the 12 constructs and the control Fc molecule DRA222 (TSC311 or TSC312) are shown in Table 1. 14.

Table 3 Variable domain composition of initial constructs

heavy chain light chain L1 (original light chain) L4 L5 H7 H7L1 (TSC313) H7L4 (TSC314) H7L5 (TSC315) H8 H8L1 (TSC316) H8L4 (TSC317) H8L5 (TSC318) H9 H9L1 (TSC319) H9L4 (TSC320) H9L5 (TSC321) NUDE H10L1 (TSC322) H10L4 (TSC323) H10L5 (TSC324)

All 12 constructs were transiently expressed in HEK293 cells, purified and tested for binding to Jurkat T cells and evaluated for thermal stability. Constructs containing the L4 light chain or the H9 heavy chain had lower levels of protein expression (see final yield column in Table 4) and/or higher levels of high molecular weight aggregates (see Analytical SEC column in Table 4). 4) and were excluded at the next stage of optimization. Although most of the other constructs showed some improvement in scFv thermal stability compared to the original humanized domains (TSC312) as measured by differential scanning calorimetry (DSC) midpoint thermal denaturation (Tm) measurements (Table 5), the level of binding saturation observed for T cells Jurkat was reduced to different levels (Fig. 1). The most stable construct, TSC324, showed an approximately 50% reduction in observed median fluorescence intensity at saturation and a twofold increase in EC ₅₀ binding (3.6 nM) compared to the original humanized construct (TSC312, also known as DRA222).

Table 4. Expression and degree of purification of transiently expressed material

Molecule Name in the database Title on 1st Finite Analytical Analytical data stage yield (mg) ex-basic EH- (µg/ml) peak (%) recovery (%)

Fc DRA222 TSC312 43.8 4.12 92.6 100.3 FCH7L1 TSC313 35.2 2.66 95.3 90.2 Fc H7L4 TSC314 57.8 5.78 58.8 96.0 Fc H7L5 TSC315 52.6 6.99 90.6 93.3 FcH8Ll TSC316 39.9 3.73 93.4 99.5 Fc H8L4 TSC317 52.9 4.95 66.5 97.6 Fc H8L5 TSC318 48.1 4.83 92.1 96.2 Fc DRA222 TSC312 16.1 1.41 95.4 97.8 FcH9Ll TSC319 8.0 0.71 80.0 85.8 Fc H9L4 TSC320 43.1 3.60 69.3 102.5 Fc H9L5 TSC321 31.1 2.66 92.6 98.6 Fc H10L1 TSC322 45.6 3.94 73.2 56.7 Fc H10L4 TSC323 50.6 4.58 67.3 106.7 Fc H10L5 TSC324 33.1 2.85 90.7 104.0

Table 5. Thermal stability determined by differential scanning calorimetry

molecules Tm anti-POPs scFv (°C) Improvement in Tm (compared to DRA222) TSC312(DRA222) 53.02 TSC313(H7L1) 54.47 1.45 TSC315(H7L5) 54.99 1.97 TSC316(H8L1) 53.70 0.68 TSC318 (H8L5) 55.16 2.14 TSC312(H7L1) 52.88 -0.14 TSC321 (H9L5) 53.10 0.08 TSC324(H10L5) 55.83 2.81

Stage 2.

Initial optimization to re-bind to CD3.

The aim of the next step was to improve binding to CD3 while maintaining improved thermal stability compared to DRA222. At this stage, three additional light chain sequences were introduced. The first light chain was based on the L5 sequence containing two amino acids changed back to the parent mouse residues at positions 52 and 53 (LL to RW) and this light chain was named L6. Also used were two additional germline light chains (IGKV1-39*01 (L7) and IGKV3D-20*1 (L8) containing the same two reverse amino acids at positions 52 and 53. 3 new light chains (L6, L7 and L8 ) was combined with 3 heavy chains (H7, H8 and H10) in anti-CD3 scFv Fc format to generate the following scFv combinations (Table 6).

- 42 042409

Table 6 Variable domain composition of initial constructs

heavy chain light chain L6 L7 L8 H7 H7L6 (TSC334) H7L7 (TSC335) H7L8 (TSC336) H8 H8L6 (TSC337) H8L7 (TSC338) H8L8 (TSC339) NUDE H10L6 (TSC340) H10L7 (TSC341) H10L8 (TSC342)

These 9 constructs were transiently expressed in HEK293 cells and examined for protein quality, expression, binding and thermostability. The sequences of these 9 designs are given in table. 14. A series of H10 molecules (TSC340, TSC341, TSC342) exhibited the best binding of all novel constructs (FIG. 2) and achieved binding saturation levels comparable to TSC312 on Jurkat T cells. However, the measured EC50 binding was still twice that of the original TSC312 molecule. The H10 series of molecules also showed a significant improvement in thermal stability as measured by the increase in Tm compared to CTSC312 by differential scanning fluorometry (FIG. 3). Of these three molecules, TSC342 was selected as the lead molecule for further optimization due to improved thermal stability.

The parent mouse sequence was then investigated for potential hotspots for introducing mutations to improve binding. Three residues were selected for reverse substitution and independently mutated back: G27Y(TSC370), M53I(TSC371) and I21M(TSC372). Binding studies of these three constructs on Jurkat T cells revealed that the G27Y mutation in the heavy chain restored CD3 binding to levels comparable to the original TSC312 construct (FIG. 4). The stability of these three constructs was also evaluated by DSC. Although the G27Y mutation did not improve Tm, surprisingly both remaining mutations, M53I in the heavy chain and 121m in the light chain, independently improved Tm by 3-4°C (Table 7).

Table 7. Thermal stability of mutants designed to improve binding

Mutations Constructions Tm (°C) ATm (°C) No mutation TSC312 52.8 - TSC342G27Y TSC370 53.6 0.8 TSC342 M53I TSC371 55.97 3.17 TSC342 I21M TSC372 56.55 3.75

Stage 3.

Final optimization step to improve thermal stability The homology model of TSC370 was examined using the susceptibility to spatial aggregation to identify hot spots for potential aggregation. One mutation, A9P in the heavy chain, was found to reduce a potential aggregation hotspot in the homology model. This mutation was introduced into the TSC370 framework to generate TSC390. This mutation alone had little effect on improving Tm (0.25°C). The A9P mutation was combined with the M53I and I21M mutations described above to generate the following anti-CDS Fc constructs (Table 8):

Table 8. Rationale for the group of mutations in the constructs of the fourth round

Structures G27Y A9P M53I I21M (improvement (improvement (improvement (improvement) affinity) stability) stability) stability) TSC390 X X TSC391 X X X TSC392 X X X TSC393 X X X TSC394 X X X X

Table 9. Thermal stability of fourth round structures as assessed by DSC

Mutations Name Tm LTT

No TSC312 mutation 52.8

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TSC370_A9P TSC390 53.05 0.25 TSC370_A9P_ M53I TSC391 56.57 3.77 TSC370_A9P_I21M TSC392 59.15 6.35 TSC370_ M53l_ I21M TSC393 58.29 5.49 TSC370_A9P_ M53l_ I21M TSC394 59.3 6.5

Combining two or more of these mutations appears to have a beneficial effect on thermal stability, as indicated by a significant increase in Tm as determined by DSC analysis (Table 9). Surprisingly, the A9P mutation also appeared to be synergistic with other mutations, providing an approximately 1 to 2.6 C increase in stability compared to the corresponding constructs without the A9P mutation. More importantly, stabilizing mutations did not adversely affect CD3 binding (FIG. 5).

Bispecific molecules targeting PSMA and CD3 have also been designed using these new anti-CD3 scFv molecules to investigate the effect, if any, of changes in anti-CD3 scFv on redirected T cell cytotoxicity (PTCC) activity. The original humanized construct (DRA222) is very effective in redirecting T cell cytotoxicity (see eg US 2014/0161800). The new constructs were investigated for their ability to exhibit similar activity. Four different anti-PSMA x anti-CD3 constructs were created using TSC391, TSC392, TSC393, and TSC394, which were named TSC408, TSC409, TSC410, and TSC411, respectively. All four constructs had similar PTKC activity to the parental DRA222 scFv (TSC249) generated molecule (FIG. 6), confirming that cytotoxic activity as well as binding were comparable to human T cells.

Stage 4.

Optimization step to recover cross-reactivity and activity for cynomolgus monkeys.

The original humanized construct (DRA222) has previously been shown to also bind cynomolgus monkey T cells and redirect their cytotoxic activity to target cells when used in a bispecific format. TSC408, TSC409, TSC410 and TSC411 were evaluated for binding and cytotoxic activity to cynomolgus monkey T cells. Surprisingly, TSC408, TSC409, TSC410, and TSC411 showed reduced binding to cynomolgus monkey T cells (FIGS. 7A and 7B) and a significant reduction in PTCC activity using cynomolgus monkey T cells as effector cells compared to anti-PSMA x anti-CD3. a bispecific molecule containing DRA222 (FIGS. 8A and 8B). This was unexpected as binding and activity with human T cells were directly comparable to constructs containing DRA222. Two approaches have been used in an attempt to restore binding to cynomolgus monkey CD3. One approach was to combine the light chain from TSC394 with the heavy chain from DRA222, or to combine the heavy chain from TSC394 with the light chain from DRA222 to see if single framework specific framework residues contribute to binding. The second approach was to back-mutate the residues at positions 86 and 87 in the TSC394 light chain. Residues at these two positions interact with the light chain CDRs, which may affect binding to cynomolgus CD3. These variants were introduced into anti-CD37 x anti-CD3 bispecific molecules (Table 10). Some of these variants, especially TSC455, TSC456, and TSC452, showed improved binding to cynomolgus monkey T cells compared to anti-CD37 x TSC394 (TSC445), as reflected by higher levels of binding at saturating concentrations (FIG. 9). An analysis of cynomolgus monkey PTCC was also performed, which showed that TSC456 and TSC452 had activity comparable to CAS105 (FIG. 10). In addition, these molecules had superior thermal stability compared to CAS105 (Table 11), as indicated by a higher Tm1 value in DSC analysis. The alignment of DRA222 scFv, TSC455 scFv and TSC456 scFv is shown in FIG. 13.

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Table 10. Fifth round of optimized designs

Anti-POP constructs Description of anti-CO37 X anti-POP bispecific molecules

CAS105 anti-CO37 x DRA222 TSC445 anti-CO37 x TSC394 TSC452 anti-СО37 x (TSC394VL+ DRA222 VH) TSC453 anti-CO37 x (TSC394VH + DRA222 VL) TSC454 anti-CO37 xTSC394 E86D TSC455 anti-CO37 x TSC394 F87Y TSC456 anti-CO37 x TSC394 E86D F87Y

Table 11. Stability of bispecific constructs

Anti-POPs designs Description of anti-CO37 X anti-POPs bispecific molecules Tm 1 CAS105 anti-CO37x DRA222 53.04 TSC445 anti-CO37 x TSC394 55.45 TSC452 anti-СО37 x (TSC394VL+ DRA222 vh) 55.37 TSC453 anti-CO37 x (TSC394VH + DRA222 VL) 56.54 TSC454 anti-CO37 x TSC394 E86D 57.94 TSC455 anti-CO37 x TSC394 F87Y 56.73 TSC456 anti-CO37x TSC394 E86D F87Y 55.45

Example 2 Effect of Improved Thermal Stability on Storage Stability.

In principle, proteins with improved thermodynamic stability should also be more resistant to aggregation after storage and should have increased storage stability compared to less stable proteins. To determine whether or not the increase observed for Tm correlates with the improvement in storage stability, the proteins shown in Table. 10 was evaluated for storage stability in PBS at 25° C. for two weeks. Each protein was buffer exchanged with PBS using preparative size exclusion chromatography and the protein concentration was adjusted to 1 mg/mL. Four vials were prepared for each protein to be evaluated, each containing approximately 120 μl. One vial was used at each stability time point. Purity was determined by assaying 25 μl (or 25 μg) on an analytical size exclusion HPLC column equilibrated in PBS and measuring absorbance at 280 nm. For each design, three repeated injections were performed at each time point. After completion of the SEC procedure, the chromatogram was integrated using the Agilent ChemStation software. The percentage purity of each protein was calculated by dividing the peak area for the intact molecule by the total peak area and then multiplying by 100.

(Peak area for intact molecule) / (total peak area) x 100 =% purity

Purity reported is an average of values obtained from three injections from one vial. Purity, as a rule, was determined at T=0, 3, 7 and 14 days. Purity values were plotted as a function of time and a linear regression analysis was performed. The slope of the regression line represented the rate of decline in purity for each protein, the rate of decline in purity was used to estimate the number of storage days that resulted in a 2% reduction in purity. The stability of different options was compared, ordering them from the largest to the smallest number of days, resulting in an estimated 2% reduction. To reduce inter-assay variability, storage stability values for different experiments were not compared, ranking was performed only for constructs within the same experimental group.

All new molecules showed superior stability in solution in PBS at 25°C compared to CAS105 (FIG. 11). Also, for each structure, the time of formation of aggregates was calculated (Table 12).

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Table 12. Relative storage stability at 25°C Anti-POPs Description of anti-CO37 X anti- # days before design CD3 bispecific 2% molecules decrease CAS105 AHTM-CD37 x DRA222 4 TSC445 AHTM-CD37 x TSC394 8 TSC450 AHTM-CD37 x TSC313 12 TSC451 AHTM-CD37 x TSC316 12 TSC452 AHTM-CD37 x (TSC394VL+H4) 7 TSC453 AHTM-CD37 x (TSC394VH+L1) 8 TSC454 AHTM-CD37 x TSC394 E462D 7 TSC455 AHTM-CD37 x TSC394 F463Y 10 TSC456 AHTM-CD37 x TSC394 DY 10

These data show that nearly all constructs had a twofold or greater increase in storage stability at 25°C compared to the construct containing the original anti-CD3 scFv (DRA222).

Example 3 Effect of Improved Thermal Stability on Serum Stability.

Similar to storage stability, molecules with higher thermodynamic stability are also often more resistant to proteolysis, which can improve stability in human serum. This, in turn, may improve the overall serum pharmacokinetics and the overall exposure of the therapeutic agent.

To investigate whether the improvement in thermodynamic stability affected overall serum stability, one of the stabilized anti-CD3 scFv molecules (TSC394 F463Y) was evaluated for serum stability in the context of an anti-ROR1 x anti-CD3 bispecific molecule (ROR193). A similar bispecific molecule was also simultaneously evaluated, which contained the original anti-CD3 scFv, DRA222 (ROR133). The parental rabbit anti-ROR1 antibody R11 used to create RORi binding domains is described, for example, in US Patent Application Publication No. 2013/0251642 and Yang et al., PLoS ONE 6(6): e21018 (2011). Human serum obtained from a random healthy donor was collected in Red/Grey Vacutanor (BD# 367988) and prepared according to the protocol suggested by the supplier. The test preparations were added to 50 µl of serum at a concentration of 1 µM in sterile PCR tubes and incubated in a humid tissue culture incubator at 37°C for up to 21 days. The selected time points were 21, 14, 7, 3 and 0 days. Samples were incubated in reverse chronological order starting from day 21 of analysis, with all samples evaluated simultaneously using the PTCC assay based on chromium release at the end of incubation on day 0 of the experiment, following the protocol given above in Example 1. EC values ₅₀ were approximated from the titration curves that were performed on the samples for each time point and normalized to the EC ₅₀ value determined for each construct at day 0.

Plotted EC ₅₀ values over time showed a huge difference in observed serum stability of ROR133 compared to ROR193 (FIG. 12) with a 2.5-fold decrease in observed EC ₅₀ over 21 days for ROR133 but minimal change in EC ₅₀ for ROR193. This demonstrates that a moderate change in thermodynamic stability can have a marked effect on serum stability.

Example 4 Effect of Improved Thermodynamic Stability on Protein Expression and Quality.

It has also been previously shown that improved thermodynamic stability can lead to improved protein expression and overall protein quality as measured by high molecular weight aggregate formation during protein production.

To investigate whether or not the improved thermodynamic stability of novel anti-CD3 scFv regions resulted in improved protein expression or protein quality, one of the anti-CD3 domains (TSC394DY) was compared to DRA222 in the context of five different pairs of anti-ROR1 x anti-CD3 bispecific molecules, each which contained the same anti-ROR1 scFv (Table 13).

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Table 13 Relative Expression and Protein Quality %

Couple sd ID designs CD3 binding domain Expression (µg/ml) % improvements EH (postproA) % wm aggregates reduction of aggregates A ROR134 DRA222 26 78 22 ROR189 TSC394DY 37 42% 89 eleven 50% IN ROR154 DRA222 16 91 9 ROR185 TSC394DY 26 63% 94 6 33% WITH ROR179 DRA222 14 83 17 ROR186 TSC394DY 27 93% 88 12 29% D R0R181 DRA222 25 81 19 R0R191 TSC394DY 33 32% 90 10 47% E ROR182 DRA222 16 90 10 ROR192 TSC394DY 21 31% 92 8 20%

For each pair of molecules, a higher titer of total protein expression was observed - from 31% to 93% higher - in the case of a construct containing a stabilized anti-CD3 scFv (TSC394DY). Also, within each molecule pair, the construct containing the stabilized antu-CD3 scFv had a lower level of high molecular weight aggregates after purification with protein A (approximately 20 to 50% reduction in aggregate levels). This confirms that incorporation of a stabilized anti-CD3 scFv can result in improved protein expression and improved protein quality compared to the original anti-CD3 scFv.

Example 5 Effect of Stabilized Anti-CD3 Binding Domains on the Stability and Pharmacokinetics of Anti-PSMA X Anti-CD3 Binding Molecules.

To investigate the effect of stabilized anti-CD3 scFv on the stability and pharmacokinetics of the bispecific binding molecule, the PSMA binding domain of TSC266 (anti-PSMA X antiCD3 bispecific molecule containing the DRA222 CD3 binding domain) was transferred into the bispecific molecule using TSC456 anti-CD3 scFv. This new bispecific molecule was named TSC471. BALB/c mice were intravenously dosed with TSC266 and TSC471 at approximately 10 mg/kg. TSC266 was diluted in PBS while TSC471 was diluted in the formulation buffer used for all dilutions (5 mM succinate, 6.5% sucrose, 0.02% Tween 80, pH 4.8). Serum was obtained from 3 animals for 10 time points (total n = 30). The time points corresponded to 15 minutes and 2, 6, 24, 48, 72, 96, 168, 336, and 504 hours after administration of the bispecific molecules. Terminal blood sampling was used to collect a larger volume. Serum concentrations were determined using anti-PSMA binding domain capture ELISA techniques and anti-CD3 binding domain detection. Serum concentrations over time were used to determine estimated pharmacokinetic (PK) parameters using non-compartmental analysis (NCA) and compartmental analysis. Serum samples from late time points were also tested for anti-drug antibodies using standard bridging ELISA with the appropriate +/- biotin bispecific molecules. More specifically, the following ELISA methods were used. The concentrations of anti-PSMA X anti-CD3 bispecific molecules were determined using 96-well plates coated with mouse monoclonal antibody (mAb 1H5) to capture the anti-PSMA portion of each construct. The other ends of the constructs were detected using a biotin-conjugated mouse monoclonal antibody targeting the anti-CD3 binding domain (mAb 5H5), thus only intact protein can be detected by this ELISA method. Polymerized horseradish peroxidase (poly-HRP) and peroxidase fluorogenic substrate were used to quantify bound immune complexes from serum samples and analytical controls, and the results were obtained using a fluorescent plate reader. The standard curves used to calculate serum concentrations consisted of various known concentrations of the corresponding PSMA bispecific construct spiked into the ELISA diluent. SOFTMAX® Pro software was used to calculate serum concentrations using a 4-parameter logistic equation, as well as accuracy and validity for standard and test samples.

The results of these studies are shown in Fig. 14 and 15. According to non-compartmental analysis (NCA) performed with Phoenix-64 WinNonLin software, TSC471 had an approximately 2-fold improvement in half-life, along with a 3-4-fold reduction in the elimination parameter compared to TSC266. Fc TSC471 retained the functionality of the TSC266

- 47 042409 (no ADCC or CDC activity, equivalent FcRn binding) with improved stability. Fc TSC471 contains an improved stability version of the CH2 domain. The mutations are similar to those in the 234-236 region of the molecule in Fc TSC266, but Fc TSC471 contains fewer mutations in the CDC region, retaining only the K322A mutation instead of all three in the null Fc CH2. Ahtu-CD3 scFv TSC471 retained binding and activity against human and cynomolgus T cells. The linker used in TSC471 had increased resistance to CHO proteases and other PTMs and did not contain an N-linked glycosylation site.

Example 6 Effect of Stabilized Anti-CD3 Binding Domains on the Pharmacokinetics of Anti-ROR1 X Anti-CD3 Binding Molecules.

The pharmacokinetics of anti-ROR1 x anti-CD3 bispecific molecules containing the less stable DRA222 CD3 binding domain or the more stable TSC456 CD3 binding domain were compared. The following designs were evaluated. The sequence of structures are given in table. 14.

Table 14

Design Anti-RORI Anti-POPs ROR206 (CHO line: ROR206a) Binding domain A DRA222 ROR207 (CHO line: ROR207a) Binding domain A TSC456 ROR208 (CHO Line: ROR208a) Binding domain B DRA222 ROR209 (CHO line: ROR209a) Binding domain B TSC456

NSG mice were intravenously dosed with anti-ROR1 X anti-CD3 bispecific antibodies at approximately 10 mg/kg. All bispecific antibodies were diluted in PBS. Serum was obtained from 3 animals for 10 time points (n=30 for each construct) after administration and also at one time point before dosing. The time points corresponded to 15 minutes and 2, 6, 24, 48, 72, 96, 168, 336, and 504 hours after administration of the bispecific molecules. Terminal blood sampling was used to collect a larger volume. Serum concentrations were determined by ELISA methods to identify the intact molecule. Serum concentrations over time were used to determine estimated PK parameters using non-compartmental analysis (NCA) and compartmental analysis.

More specifically, the following ELISA methods were used. Concentrations of anti-ROR1 X antu-CD3 bispecific molecules were determined using 96-well plates coated with ROR1 ECD-AFH (ROR177) to capture the anti-ROR1 portion of each construct. The other ends of the ROR constructs were detected using a biotin-conjugated mouse monoclonal antibody targeting the anti-CD3 binding domain (mAb 5H5), thus only intact protein can be detected by this ELISA method. Polymerized horseradish peroxidase (poly-HRP) and peroxidase fluorogenic substrate were used to quantify bound immune complexes from serum samples and analytical controls, and the results were obtained using a fluorescent plate reader. The standard curves used to calculate serum concentrations consisted of various known concentrations of the appropriate ROR bispecific construct spiked into the ELISA diluent. SOFTMAX® Pro software was used to calculate serum concentrations using a 4-parameter logistic equation, as well as accuracy and validity for standard and test samples.

The results of these studies are shown in Fig. 16, 17A and 17B. According to NCA and compartmental analysis, ROR209a had the longest half-life. ROR207a had the lowest clearance and estimated volume in both assays. Both anti-ROR1 bispecific molecules with the DRA222 CD3 binding domain had shorter half-lives and faster clearance parameters. It is worth noting that constructs with an improved anti-CD3 binding domain (TSC456) had better pharmacokinetics than constructs with a less stable anti-CD3 binding domain (DRA222).

Example 7 Effect of Stabilized Anti-CD3 Binding Domains on In Vivo Efficacy of Anti-ROR1 X Anti-CD3 Binding Molecules.

MDA-MB-231 cells were mixed together with donor T cells and Matrigel and implanted into the flank of NOD/SCID mice on day 0 of the study. Each group contained N = 5 animals with T cells from one donor. In FIG. 18 shows a typical graph that demonstrates the minimal effect of T cells on tumor growth from donor T cells. Animals were treated with PBS or 30 µg or 3 µg of ROR208 (anti-CD3 binding domain of DRA222) or ROR209 (anti-CD3 binding

- 48 042409 TSC456 domain). The dose was administered on days 0, 4 and 8. Tumor growth was measured with calipers throughout the study.

Significant tumor growth inhibition was observed after treatment with both bispecific ROR molecules. No significant difference in tumor growth was observed with T cells from the donor (FIG. 18) compared with the untreated tumor. No significant difference was observed between ROR208-treated animals compared to ROR209-treated animals at any dosage level, indicating that the anti-CD3 binding domain with improved stability was as effective as the unstable anti-CD3 binding domain. Example 8 Effect of Stabilized Anti-CD3 Binding Domains on In Vivo Efficacy of Anti-ROR1 X Anti-CD3 Binding Molecules Kasumi-2 cells were mixed together with donor T cells and Matrigel and implanted in the flank of NOD/SCID mice on day 0 of the study. Each group contained N = 10 animals with T cells from one donor. Animals were treated with PBS or 30, 3, or 0.3 μg of ROR243 (anti-CD3 binding domain of TSC456) as shown in FIG. 19. The dose was administered on days 0, 4 and 8. The route of administration was intravenous (IV) with the exception of one group in which the dose was administered subcutaneously (S/C). In FIG. 19 shows the results of the analysis. Tumor growth was measured with calipers throughout the study.

Tumor growth inhibition was not observed in the presence of T cells in the absence of ROR243 or with ROR243 treatment in the absence of T cells. Significant inhibition of tumor growth was observed using a dose of 0.3 μg with a dose-dependent titration.

No difference was observed between routes of administration.

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Table 14 Binding domain and polypeptide sequences and components

DNA sequence for Fc DRA222 (TSC311 or TSC312):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt ggagtctggg840 ggcggagtgg tgcagcctgg gcggtcactg ag gctgtcct gcaaggcttc tggctacacc900 tttactagat ctacgatgca ctgggtaagg caggcccctg gacaaggtct ggaatggatt960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacaggttc1020 acaatcagcg cagacaaatc caagagcaca gccttcctgc agatggacag cctgaggccc1080 gaggacaccg gcgtctattt ctgtgcacgg ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaagggac tcccgtcact gtctctagcg gtggcggagg gtctgggggt1200 ggcggatccg gaggtggtgg ctctgcacaa gacatccaga tgacccagtc tccaagcagc1260 ctgtctgcaa gcgtggggga cagggtcacc atgacctgca gtgccagctc aagtgtaagt1320 tacatgaact ggtaccagca gaagccgggc aaggccccca aaagatggat ttatgactca1380 tccaaactgg cttctggagt ccctgctcgc ttcagtggca gtgggtctgg gaccgactat1440 accctcacaa tcagcagcct gcagcccgaa gatttcgcca cttattactg ccagcagtgg1500 agtcgtaacc cacccacgtt cggagggggg accatacctac aacat kaccactac5 (ID01)

The signal sequence is represented by residues 1 to 60. The Fc region is represented by residues 61-753. The linker is represented by residues 754 to 816. Anti-SVZ scFv is represented by residues 817 to 1560.

Mature protein sequence for Fc DRA222 (TSC311 or TSC312)

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVESG GGWQPGRSL RLSCKASGYT FTRSTMHWVR QAPGQGLEWI300

GYINPSSAYT NYNQKFKDRF TISADKSKST AFLQMDSLRP EDTGVYFCAR PQVHYDYNGF360

PYWGQGTPVT VSSGGGGSGG GGSGGGGSAQ DIQMTQSPSS LSASVGDRVT MTCSASSSVS420

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YMNWYQQKPG KAPKRWIYDS SKLASGVPAR FSGSGSGTDY TLTISSLQPE DFATYYCQQW480

SRNPPTFGGG TKLQITSSS499 (SEQ ID NO:2)

DNA sequence for Fc H7L1 (TSC313):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccaagcagcc tgtctgcaag cgtgggggac agggtcacca tgacctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aagccgggca aggcccccaa aagatggatt1380 tatgactcat ccaaactggc ttctggagtc cctgctcgct tcagtggcag tgggtctggg1440 accgactata ccctcacaat cagcagcctg cagcccgaag atttcgccac ttattactgc1500 cagcagtgga gtcgtaaccc acccacgttc ggagggggga ccaagctaca aattacatcc1560 tccagctaa1569 (SEQ ID NO:3)

The signal sequence is represented by residues 1 to 60. The Fc region is represented by residues 61-753. The linker is represented by residues 754 to 816. Anti-SVZ scFv is represented by residues 817 to 1569.

Mature protein sequence for Fc H7L1 (TSC313):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

- 51 042409

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PARFSGSGSG TDYTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKLQITS SS502 (SEQ ID NO:4)

DNA sequence for Fc H7L4 (TSC314):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacacagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccaacag aaacctggcc aggctcccag gctcctcatc1380 tatgactcat ccaaactggc ttctggcatc ccagccaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:5)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H7L4 (TSC314):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGQAPRLLI YDSSKLASGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC480

- 52 042409

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:6)

DNA sequence for Fc H7L5 (TSC315):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gctcctgatc1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:7)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H7L5 (TSC315):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKLLI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503

- 53 042409 (SEQ ID NO:8)

DNA sequence for Fc H8L1 (TSC316):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccaagcagcc tgtctgcaag cgtgggggac agggtcacca tgacctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aagccgggca aggcccccaa aagatggatt1380 tatgactcat ccaaactggc ttctggagtc cctgctcgct tcagtggcag tgggtctggg1440 accgactata ccctcacaat cagcagcctg cagcccgaag atttcgccac ttattactgc1500 cagcagtgga gtcgtaaccc acccacgttc ggagggggga ccaagctaca aattacatcc1560 tccagctaa1569 (SEQ ID NO:9)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L1 (TSC316):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PARFSGSGSG TDYTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKLQITS S3502 (SEQIDNO:10)

- 54 042409

DNA sequence for Fc H8L4 (TSC317):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacacagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccaacag aaacctggcc aggctcccag gctcctcatc1380 tatgactcat ccaaactggc ttctggcatc ccagccaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:11)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L4 (TSC317):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGQAPRLLI YDSSKLASGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQIDNO:12)

- 55 042409

DNA sequence for Fc H8L5 (TSC318):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gctcctgatc1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:13)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L5 (TSC318):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT12 0

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKLLI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQIDNO:14)

- 56 042409

DNA sequence for Fc H9L1 (TSC319):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagcttgt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcttc tggctacacc900 tttactagat ctacgatgca ttgggtgcgc caggcccccg gacaaaggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accattacca gggacacatc cgcgagcaca gcctacatgg agctgagcag cctgagatct1080 gaagacacgg ctgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccaagcagcc tgtctgcaag cgtgggggac agggtcacca tgacctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aagccgggca aggcccccaa aagatggatt1380 tatgactcat ccaaactggc ttctggagtc cctgctcgct tcagtggcag tgggtctggg1440 accgactata ccctcacaat cagcagcctg cagcccgaag atttcgccac ttattactgc1500 cagcagtgga gtcgtaaccc acccacgttc ggagggggga ccaagctaca aattacatcc1560 tccagctaa1569 (SEQ ID NO: 15)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H9L1 (TSC319):

EPKSSDKTHT CPPCPAPEAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF 60 NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT 120 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN 240 TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQRLEWM 300 GYINPSSAYT NYNQKFKDRV TITRDTSAST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF 360 PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTMTCSASS 420 SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PARFSGSGSG TDYTLTISSL QPEDFATYYC 480 QQWSRNPPTF GGGTKLQITS SS 502

(SEQ ID NO: 16)

- 57 042409

DNA sequence for Fc H9L4 (TSC320):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagcttgt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcttc tggctacacc900 tttactagat ctacgatgca ttgggtgcgc caggcccccg gacaaaggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accattacca gggacacatc cgcgagcaca gcctacatgg agctgagcag cctgagatct1080 gaagacacgg ctgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacacagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccaacag aaacctggcc aggctcccag gctcctcatc1380 tatgactcat ccaaactggc ttctggcatc ccagccaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO: 17)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H9L4 (TSC320):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQRLEWM300

GYINPSSAYT NYNQKFKDRV TITRDTSAST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGQAPRLLI YDSSKLASGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQIDNO:18)

- 58 042409

DNA sequence for Fc H9L5 (TSC321):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagcttgt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcttc tggctacacc900 tttactagat ctacgatgca ttgggtgcgc caggcccccg gacaaaggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accattacca gggacacatc cgcgagcaca gcctacatgg agctgagcag cctgagatct1080 gaagacacgg ctgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gctcctgatc1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO: 19)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H9L5 (TSC321):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQRLEWM300

GYINPSSAYT NYNQKFKDRV TITRDTSAST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKLLI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:20)

DNA sequence for Fc H10L1 (TSC322):

- 59 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg g tcctcggtg aaggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccaagcagcc tgtctgcaag cgtgggggac agggtcacca tgacctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aagccgggca aggcccccaa aagatggatt1380 tatgactcat ccaaactggc ttctggagtc cctgctcgct tcagtggcag tgggtctggg1440 accgactata ccctcacaat cagcagcctg cagcccgaag atttcgccac ttattactgc1500 cagcagtgga gtcgtaaccc acccacgttc ggagggggga ccaagctaca aattacatcc1560 tccagctaa1569 (SEQ ID NO:21)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for FC H10L1 (TSC322):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PARFSGSGSG TDYTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKLQITS S3502 (SEQ ID NO:22)

DNA sequence for Fc H10L4 (TSC323)

- 60 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg g tcctcggtg aaggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacacagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccaacag aaacctggcc aggctcccag gctcctcatc1380 tatgactcat ccaaactggc ttctggcatc ccagccaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagccta gagcctgaag attttgcagt ttattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:23)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H10L4 (TSC323)

EPKSSDKTHT CPPCPAPEAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF 60 NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT 120 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN 240 TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM 300 GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF 360 PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS 420 SVSYMNWYQQ KPGQAPRLLI YDSSKLASGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC 480 QQWSRNPPTF GGGTKVEIKR SSS 503

(SEQ ID NO:24)

- 61 042409

DNA sequence for Fc H10L5 (TSC324):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gctcctgatc1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:25)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H10L5 (TSC324):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKLLI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:26)

DNA sequence for Fc H7L6 (TSC334):

- 62 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg g gcctcagtg aaggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:27)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H7L6 (TSC334):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS*504 (SEQ ID NO:28)

DNA sequence for Fc H7L7 (TSC335):

- 63 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg g gcctcagtg aaggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacgcagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aaacctggcc tggcgcccag gagatggatt1380 tatgactcat ccaaactggc ttctggcatc ccagacaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagactg gagcctgaag attttgcagt gtattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:29)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H7L7 (TSC335):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGLAPRRWI YDSSKLASGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC480

QQWSRNPPTF GGGTKVEIKR SSS504 (SEQ ID Νθ:30)

DNA sequence for Fc H7L8 (TSC336):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60

- 64 042409 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aaggtctcct gcaaggcttc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagggtc1020 accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct1080 gacgacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt (SEQ ID NO:31)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H7L8 (TSC336):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSIST AYMELSRLRS DDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS*504 (SEQ ID NO:32)

DNA sequence for Fc H8L6 (TSC337)

- 65 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg g gcctcagtg aaggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:33)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L6 (TSC337):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS*504 (SEQ ID NO:34)

DNA sequence for Fc H8L7 (TSC338):

- 66 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg g gcctcagtg aaggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacgcagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aaacctggcc tggcgcccag gagatggatt1380 tatgactcat ccaaactggc ttctggcatc ccagacaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagactg gagcctgaag attttgcagt gtattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:35)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L7 (TSC338):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGLAPRRWI YDSSKLASGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:36)

- 67 042409

DNA sequence for Fc H8L8 (TSC339):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tgcagctggt gcagtctggg840 gctgaggtga agaagcctgg ggcctcagtg aa ggtttcct gcaaggcatc tggatacacc900 ttcaccagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 accatgacca gggacacgtc cacgagcaca gtctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgctaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagaattca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:37)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H8L8 (TSC339):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGASV KVSCKASGYT FTRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TMTRDTSTST VYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS504 (SEQ ID NO:38)

- 68 042409

DNA sequence for Fc H10L6 (TSC340):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagtggcag tggatctggg1440 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:39)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H10L6 (TSC340):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:40)

DNA sequence for Fc H10L7 (TSC341):

- 69 042409 atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg g tcctcggtg aaggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg aaattgtgtt gacgcagtct1260 ccagccaccc tgtctttgtc tccaggggaa agagccaccc tctcctgcag tgccagctca1320 agtgtaagtt acatgaactg gtaccagcag aaacctggcc tggcgcccag gagatggatt1380 tatgactcat ccaaactggc ttctggcatc ccagacaggt tcagtggcag tgggtctggg1440 acagacttca ctctcaccat cagcagactg gagcctgaag attttgcagt gtattactgt1500 cagcagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:41)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H10L7 (TSC341):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSEIVLTQS PATLSLSPGE RATLSCSASS420

SVSYMNWYQQ KPGLAPRRWI YDSSKLASGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:42)

- 70 042409

DNA sequence for Fc H10L8 (TSC342):

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagaattca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:43)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for Fc H10L8 (TSC342):

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:44)

- 71 042409

DNA sequence for TSC370:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagaattca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:45)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC370:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:46)

- 72 042409

DNA sequence for TSC371:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggata960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagaattca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:47)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC371:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF 60 NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT 120 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN 240 TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWI 300 GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF 360 PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS 420 SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC 480 QQWSRNPPTF GGGTKVEIKR SSS 503

- 73 042409 (SEQ ID NO:48)

DNA sequence for TSC372:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggaggcacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tgacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagaattca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:49)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC372:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGGT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503

- 74 042409 (SEQ ID NO:50)

DNA sequence for TSC390:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 cctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagagttca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:51)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC390:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCWVD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG PEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503

- 75 042409 (SEQ ID NO:52)

DNA sequence for TSC391:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 cctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggata960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagagttca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:53)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC391:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG PEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWI300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTITCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

QQWSRNPPTF GGGTKVEIKR SSS503

- 76 042409 (SEQ ID NO:54)

DNA sequence for TSC392:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 cctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggatg960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tgacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagagttca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:55)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC392:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG PEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWM300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

- 77 042409

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:56)

DNA sequence for TSC393:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 gctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggata960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tgacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagagttca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:57)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC393:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG AEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWI300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

- 78 042409

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:58)

DNA sequence for TSC394:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg120 ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg180 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc240 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag300 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat360 ggcaaggcat acgcatgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc420 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg480 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc540 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct600 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc660 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac720 tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat780 acaggaactc agatggcagg tcattctccg aattctcagg tccagctggt gcaatctggg840 cctgaggtga agaagcctgg gtcctcggtg aa ggtctcct gcaaggcttc tggatatacc900 ttcagcagat ctacgatgca ctgggtgcga caggcccctg gacaagggct tgagtggata960 ggatacatta atcctagcag tgcttatact aattacaatc agaaattcaa ggacagagtc1020 acgattaccg cggacaaatc cacgagcaca gcctacatgg agctgagcag cctgagatct1080 gaggacacgg ccgtgtatta ctgtgcgaga ccccaagtcc actatgatta caacgggttt1140 ccttactggg gccaaggaac cctggtcacc gtctcctcag gtggaggcgg ttcaggcgga1200 ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatccagat gacccagtct1260 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tgacttgcag tgccagctca1320 agtgtaagtt acatgaactg gtatcagcag aaaccaggga aagcccctaa gagatggatt1380 tatgactcat ccaaactggc ttctggggtc ccatcaaggt tcagcggcag tggatctggg1440 acagagttca ctctcaccat cagcagcctg cagcctgatg attttgcaac ttattactgc1500 caacagtgga gtcgtaaccc acccactttc ggcggaggga ccaaggtgga gatcaaacgg1560 tcctccagct aa1572 (SEQ ID NO:59)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC394:

EPKSSDKTHT SRRSRAREAAA GAPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF60

NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKAYACAVSN KALPAPIEKT120

ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP180

PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GQRHNNSSLN240

TGTQMAGHSP NSQVQLVQSG PEVKKPGSSV KVSCKASGYT FSRSTMHWVR QAPGQGLEWI300

GYINPSSAYT NYNQKFKDRV TITADKSTST AYMELSSLRS EDTAVYYCAR PQVHYDYNGF360

PYWGQGTLVT VSSGGGGSGG GGSGGGGSGG GGSDIQMTQS PSTLSASVGD RVTMTCSASS420

SVSYMNWYQQ KPGKAPKRWI YDSSKLASGV PSRFSGSGSG TEFTLTISSL QPDDFATYYC480

- 79 042409

QQWSRNPPTF GGGTKVEIKR SSS503 (SEQ ID NO:60)

DNA sequence for TSC408:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gatatccaga tgacccagtc tccatccgcc atgtctgcat ctgtaggaga cagagtcacc120 atcacttgcc gggcgagtaa gagcattagc aaatatttag cctggtttca gcagaaacca180 gggaaagttc ctaagctccg catccattct ggatctactt tgcaatcagg ggtcccatct240 cggttcagtg gcagtggatc tgggacagaa tttactctca ccatcagcag cctgcagcct300 gaagattttg caacttatta ctgtcaacag catattgaat acccgtggac gttcggccaa360 gggaccaagg tggaaatcaa acgaggtggc ggagggtctg ggggtggcgg atccggaggt420 ggtggctctc aggtccagct ggtacagtct ggggctgagg tgaagaagcc tggggcttca480 gtgaaggtct cctgcaaggc ttctggatac acattcactg actactacat gcactgggtg540 cgacaggccc ctggacaagg gcttgagtgg atgggatatt ttaatcctta taatgattat600 actagatacg cacagaagtt ccagggcaga gtcaccatga ccagggacac gtctatcagc660 acagcctaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca720 agatcggatg gttactacga tgctatggac tactggggtc aaggaaccac agtcaccgtc780 tcctcgagtg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct840 gaagccgcgg gtgcaccgtc agtcttcctc tt ccccccaa aacccaagga caccctcatg900 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag960 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg1020 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac1080 tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc1140 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc1200 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc1260 tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag1320 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg1380 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg1440 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtcagaggca caacaattct1500 tccctgaata caggaactca gatggcaggt cattctccga attctcaggt ccagctggtg1560 caatctgggc ctgaggtgaa gaagcctggg tcctcggtga aggtctcctg caaggcttct1620 ggatatacct tcagcagatc tacgatgcac tgggtgcgac aggcccctgg acaagggctt1680 gagtggatag gatacattaa tcctagcagt gcttatacta attacaatca ga aattcaag1740 gacagagtca cgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagc1800 ctgagatctg aggacacggc cgtgtattac tgtgcgagac cccaagtcca ctatgattac1860 aacgggtttc cttactgggg ccaaggaacc ctggtcaccg tctcctcagg tggaggcggt1920 tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catccagatg1980 acccagtctc cttccaccct gtctgcatct gtaggagaca gagtcaccat cacttgcagt2040 gccagctcaa gtgtaagtta catgaactgg tatcagcaga aaccagggaa agcccctaag2100 agatggattt atgactcatc caaactggct tctggggtcc catcaaggtt cagcggcagt2160 ggatctggga cagagttcac tctcaccatc agcagcctgc agcctgatga ttttgcaact2220 tattactgcc aacagtggag tcgtaaccca cccactttcg gcggagggac caaggtggag2280 atcaaacggt cctccagcta a2301 (SEQ ID NO:61)

The signal sequence is represented by residues from 1 to 60

- 80 042409

Mature protein sequence for TSC408:

DIQMTQSPSA RFSGSGSGTE GGSQVQLVQS TRYAQKFQGR SSSEPKSSDK VKFNWYVDGV EKTISKAKGQ TTPPVLDSDG SLNTGTQMAG EWIGYINPSS NGFPYWGQGT ASSSVSYMNW

MSASVGDRVT FTLTISSLQP GAEVKKPGAS VTMTRDTSIS TNTSRRSRAP EVHNAKTKPR PREPQVYTLP SFFLYSKLTV HSPNSQVQLV AYTNYNQKFK LVTVSSGGGG YQQKPGKAPK

ITCRASKSIS EDFATYYCQQ VKVSCKASGY TAYMELSSLR EAAGAPSVFL EEQYNSTYRV PSRDELTKNQ DKSRWQQGNV QSGPEVKKPG DRVTITADKS SGGGGSGGGG RWIYDSSKLA

KYLAWFQQKP

HIEYPWTFGQ

TFTDYYMHWV

SDDTAVYYCA

FPPKPKDTLM

VSVLTVLHQD

VSLTCLVKGF

FSCSVMHEAL

SSVKVSCKAS

TSTAYMELSS

SGGGGSDIQM

SGVPSRFSGS

GKVPKLRIHS GTKVEIKRGG RQAPGQGLEW RSDGYYDAMD ISRTPEVTCV WLNGKAYACA YPSDIAVEWE HNHYTQKSLS GYTFSRSTMH LRSEDTAVYY TQSPSTLSAS GSGTEFTLTI

GSTLQSGVPS GGSGGGGSGG MGYFNPYNDY YWGQGTTVTV WDVSHEDPE VSNKALPAPI SNGQPENNYK LSPGQRHNNS WVRQAPGQGL CARPQVHYDY VGDRVTITCS SSLQPDDFAT

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YYCQQWSRNP PTFGGGTKVE IKRSSS (SEQ ID NO:62)

DNA sequence for TSC409:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt gatatccaga tgacccagtc tccatccgcc atgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcgagtaa gagcattagc aaatatttag cctggtttca gcagaaacca gggaaagttc ctaagctccg catccattct ggatctactt tgcaatcagg ggtcccatct cggttcagtg gcagtggatc tgggacagaa tttactctca ccatcagcag cctgcagcct gaagattttg caacttatta ctgtcaacag catattgaat acccgtggac gttcggccaa gggaccaagg tggaaatcaa acgaggtggc ggagggtctg ggggtggcgg atccggaggt ggtggctctc aggtccagct ggtacagtct ggggctgagg tgaagaagcc tggggcttca gtgaaggtct cctgcaaggc ttctggatac acattcactg actactacat gcactgggtg cgacaggccc ctggacaagg gcttgagtgg atgggatatt ttaatcctta taatgattat actagatacg cacagaagtt ccagggcaga gtcaccatga ccagggacac gtctatcagc acagcctaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca agatcggatg gttactacga tgctatggac tactggggtc aaggaaccac agtcaccgtc tcctcgagtg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct gaagccgcgg gtgcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg

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- 81 042409 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtcagaggca caacaattct1500 tccctgaata caggaactca gatggcaggt cattctccga attctcaggt ccagctggtg1560 caatctgggc ctgaggtgaa gaagcctggg tcctcggtga aggtctcctg caaggcttct1620 ggatatacct tcagcagatc tacgatgcac tgggtgcgac aggcccctgg acaagggctt1680 gagtggatgg gatacattaa tcctagcagt gcttatacta attacaatca gaaattcaag1740 gacagagtca cgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagc1800 ctgagatctg aggacacggc cgtgtattac tgtgcgagac cccaagtcca ctatgattac1860 aacgggtttc cttactgggg ccaaggaacc ctggtcaccg tctcctcagg tggaggcggt1920 tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catccagatg1980 acccagtctc cttccaccct gtctgcatct gtaggagaca gagtcaccat gacttgcagt2040 gccagctcaa gtgtaagtta catgaactgg tatcagcaga aaccagggaa agcccctaag2100 agatggattt atgactcatc caaactggct tctggggtcc catcaaggtt cagcggcagt2160 ggatctggga cagagttcac tctcaccatc agcagcctgc agcctgatga ttttgcaact2220 tattactgcc aacagtggag tcgtaaccca cccactttcg gcggagggac caaggtggag2280 atcaaacg gt cctccagcta a2301 (SEQ ID NO:63)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC409:

DIQMTQSPSA RFSGSGSGTE GGSQVQLVQS TRYAQKFQGR SSSEPKSSDK VKFNWYVDGV EKTISKAKGQ TTPPVLDSDG SLNTGTQMAG EWMGYINPSS NGFPYWGQGT ASSSVSYMNW YYCQQWSRNP

MSASVGDRVT FTLTISSLQP GAEVKKPGAS VTMTRDTSIS TNTSRRSRAP EVHNAKTKPR PREPQVYTLP SFFLYSKLTV HSPNSQVQLV AYTNYNQKFK LVTVSSGGGG YQQKPGKAPK PTFGGGTKVE

ITCRASKSIS EDFATYYCQQ VKVSCKASGY TAYMELSSLR EAAGAPSVFL EEQYNSTYRV PSRDELTKNQ DKSRWQQGNV QSGPEVKKPG DRVTITADKS SGGGGSGGGG RWIYDSSKLA IKRSSS

KYLAWFQQKP

HIEYPWTFGQ

TFTDYYMHWV

SDDTAVYYCA

FPPKPKDTLM

VSVLTVLHQD

VSLTCLVKGF

FSCSVMHEAL

SSVKVSCKAS

TSTAYMELSS

SGGGGSDIQM

SGVPSRFSGS

GKVPKLRIHS GTKVEIKRGG RQAPGQGLEW RSDGYYDAMD ISRTPEVTCV WLNGKAYACA YPSDIAVEWE HNHYTQKSLS GYTFSRSTMH LRSEDTAVYY TQSPSTLSAS GSGTEFTLTI

GSTLQSGVPS GGSGGGGSGG MGYFNPYNDY YWGQGTTVTV WDVSHEDPE VSNKALPAPI SNGQPENNYK LSPGQRHNNS WVRQAPGQGL CARPQVHYDY VGDRVTMTCS SSLQPDDFAT

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540

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660

720

746 (SEQ ID NO:64)

DNA sequence for TSC410:

atggaagcac cagcgcagct tctcttcctc gatatccaga tgacccagtc tccatccgcc atcacttgcc gggcgagtaa gagcattagc gggaaagttc ctaagctccg catccattct cggttcagtg gcagtggatc tgggacagaa gaagattttg caacttatta ctgtcaacag gggaccaagg tggaaatcaa acgaggtggc ggtggctctc aggtccagct ggtacagtct gtgaaggtct cctgcaaggc ttctggatac ctgctactct ggctcccaga taccaccggt atgtctgcat ctgtaggaga cagagtcacc aaatatttag cctggtttca gcagaaacca ggatctactt tgcaatcagg ggtcccatct tttactctca ccatcagcag cctgcagcct catattgaat acccgtggac gttcggccaa ggagggtctg ggggtggcgg atccggaggt ggggctgagg tgaagaagcc tggggcttca acattcactg actactacat gcactgggtg

120

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240

300

360

420

480

540

- 82 042409 cgacaggccc ctggacaagg gcttgagtgg atgggatatt ttaatcctta taatgattat600 actagatacg cacagaagtt ccagggcaga gtcaccatga ccagggacac gtctatcagc660 acagcctaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca720 agatcggatg gttactacga tgctatggac tactggggtc aaggaaccac agtcaccgtc780 tcctcgagtg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct840 gaagccgcgg gtgcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg900 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag960 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg1020 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac1080 tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc1140 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc1200 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc1260 tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag1320 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg1380 gacaagagca ggtg gcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg1440 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtcagaggca caacaattct1500 tccctgaata caggaactca gatggcaggt cattctccga attctcaggt ccagctggtg1560 caatctgggg ctgaggtgaa gaagcctggg tcctcggtga aggtctcctg caaggcttct1620 ggatatacct tcagcagatc tacgatgcac tgggtgcgac aggcccctgg acaagggctt1680 gagtggatag gatacattaa tcctagcagt gcttatacta attacaatca gaaattcaag1740 gacagagtca cgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagc1800 ctgagatctg aggacacggc cgtgtattac tgtgcgagac cccaagtcca ctatgattac1860 aacgggtttc cttactgggg ccaaggaacc ctggtcaccg tctcctcagg tggaggcggt1920 tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catccagatg1980 acccagtctc cttccaccct gtctgcatct gtaggagaca gagtcaccat gacttgcagt2040 gccagctcaa gtgtaagtta catgaactgg tatcagcaga aaccagggaa agcccctaag2100 agatggattt atgactcatc caaactggct tctggggtcc catcaaggtt cagcggcagt2160 ggatctggga cagagttcac tctcaccatc agcagcctgc agcctgatga ttttgcaact2220 tattactgcc aacagtggag tcgtaaccca cc cactttcg gcggagggac caaggtggag2280 atcaaacggt cctccagcta a2301 (SEQ ID NO:65)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for TSC410:

DIQMTQSPSA MSASVGDRVT ITCRASKSIS KYLAWFQQKP GKVPKLRIHS GSTLQSGVPS60

RFSGSGSGTE FTLTISSLQP EDFATYYCQQ HIEYPWTFGQ GTKVEIKRGG GGSGGGGSGG120

GGSQVQLVQS GAEVKKPGAS VKVSCKASGY TFTDYYMHWV RQAPGQGLEW MGYFNPYNDY180

TRYAQKFQGR VTMTRDTSIS TAYMELSSLR SDDTAVYYCA RSDGYYDAMD YWGQGTTVTV240

SSSEPKSSDK TNTSRRSRAR EAAGAPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE300

VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKAYACA VSNKALPAPI360

EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK420

TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGQRHNNS480

SLNTGTQMAG HSPNSQVQLV QSGAEVKKPG SSVKVSCKAS GYTFSRSTMH WVRQAPGQGL540

EWIGYINPSS AYTNYNQKFK DRVTITADKS TSTAYMELSS LRSEDTAVYY CARPQVHYDY600

NGFPYWGQGT LVTVSSGGGG SGGGGSGGGG SGGGGSDIQM TQSPSTLSAS VGDRVTMTCS660

- 83 042409

ASSSVSYMNW YQQKPGKAPK RWIYDSSKLA SGVPSRFSGS GSGTEFTLTI SSLQPDDFAT720

YYCQQWSRNP PTFGGGTKVE IKRSSS746 (SEQ ID NO:66)

DNA sequence for TSC411:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gatatccaga tgacccagtc tccatccgcc atgtctgcat ctgtaggaga cagagtcacc120 atcacttgcc gggcgagtaa gagcattagc aaatatttag cctggtttca gcagaaacca180 gggaaagttc ctaagctccg catccattct ggatctactt tgcaatcagg ggtcccatct240 cggttcagtg gcagtggatc tgggacagaa tttactctca ccatcagcag cctgcagcct300 gaagattttg caacttatta ctgtcaacag catattgaat acccgtggac gttcggccaa360 gggaccaagg tggaaatcaa acgaggtggc ggagggtctg ggggtggcgg atccggaggt420 ggtggctctc aggtccagct ggtacagtct ggggctgagg tgaagaagcc tggggcttca480 gtgaaggtct cctgcaaggc ttctggatac acattcactg actactacat gcactgggtg540 cgacaggccc ctggacaagg gcttgagtgg atgggatatt ttaatcctta taatgattat600 actagatacg cacagaagtt ccagggcaga gtcaccatga ccagggacac gtctatcagc660 acagcctaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca720 agatcggatg gttactacga tgctatggac tactggggtc aaggaaccac agtcaccgtc780 tcctcgagtg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct840 gaagccgcgg gtgcaccgtc agtcttcctc tt ccccccaa aacccaagga caccctcatg900 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag960 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg1020 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac1080 tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc1140 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc1200 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc1260 tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag1320 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg1380 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg1440 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtcagaggca caacaattct1500 tccctgaata caggaactca gatggcaggt cattctccga attctcaggt ccagctggtg1560 caatctgggc ctgaggtgaa gaagcctggg tcctcggtga aggtctcctg caaggcttct1620 ggatatacct tcagcagatc tacgatgcac tgggtgcgac aggcccctgg acaagggctt1680 gagtggatag gatacattaa tcctagcagt gcttatacta attacaatca ga aattcaag1740 gacagagtca cgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagc1800 ctgagatctg aggacacggc cgtgtattac tgtgcgagac cccaagtcca ctatgattac1860 aacgggtttc cttactgggg ccaaggaacc ctggtcaccg tctcctcagg tggaggcggt1920 tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catccagatg1980 acccagtctc cttccaccct gtctgcatct gtaggagaca gagtcaccat gacttgcagt2040 gccagctcaa gtgtaagtta catgaactgg tatcagcaga aaccagggaa agcccctaag2100 agatggattt atgactcatc caaactggct tctggggtcc catcaaggtt cagcggcagt2160 ggatctggga cagagttcac tctcaccatc agcagcctgc agcctgatga ttttgcaact2220 tattactgcc aacagtggag tcgtaaccca cccactttcg gcggagggac caaggtggag2280 atcaaacggt cctccagcta a2301 (SEQ ID NO:67)

The signal sequence is represented by residues from 1 to 60

- 84 042409

Mature protein sequence for TSC411:

DIQMTQSPSA RFSGSGSGTE GGSQVQLVQS TRYAQKFQGR SSSEPKSSDK VKFNWYVDGV EKTISKAKGQ TTPPVLDSDG SLNTGTQMAG EWIGYINPSS NGFPYWGQGT ASSSVSYMNW YYCQQWSRNP

MSASVGDRVT FTLTISSLQP GAEVKKPGAS VTMTRDTSIS TNTSRRSRAP EVHNAKTKPR PREPQVYTLP SFFLYSKLTV HSPNSQVQLV AYTNYNQKFK LVTVSSGGGG YQQKPGKAPK PTFGGGTKVE

ITCRASKSIS EDFATYYCQQ VKVSCKASGY TAYMELSSLR EAAGAPSVFL EEQYNSTYRV PSRDELTKNQ DKSRWQQGNV QSGPEVKKPG DRVTITADKS SGGGGSGGGG RWIYDSSKLA IKRSSS

KYLAWFQQKP

HIEYPWTFGQ

TFTDYYMHWV

SDDTAVYYCA

FPPKPKDTLM

VSVLTVLHQD

VSLTCLVKGF

FSCSVMHEAL

SSVKVSCKAS

TSTAYMELSS

SGGGGSDIQM

SGVPSRFSGS

GKVPKLRIHS GTKVEIKRGG RQAPGQGLEW RSDGYYDAMD ISRTPEVTCV WLNGKAYACA YPSDIAVEWE HNHYTQKSLS GYTFSRSTMH LRSEDTAVYY TQSPSTLSAS GSGTEFTLTI

GSTLQSGVPS GGSGGGGSGG MGYFNPYNDY YWGQGTTVTV WDVSHEDPE VSNKALPAPI SNGQPENNYK LSPGQRHNNS WVRQAPGQGL CARPQVHYDY VGDRVTMTCS SSLQPDDFAT

120

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240

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360

420

480

540

600

660

720

746 (SEQ ID NO:68)

DNA sequence for CAS105:

atggaagcac gaggtgcagc tcctgtaagg cccgggaaag aaccggaagt ctgcaatgga ggccctttcg tctggtggcg tctgaaattg accctctcct cctggccagg gccaggttca cctgaagatt caagggacca acatgcccac ccaaaaccca gacgtgagcc cataatgcca gtcctcaccg aacaaagccc gaaccacagg ctgacctgcc gggcagccgg ttcctctaca tgctccgtga ccgggtcaga cagcgcagct tggtgcagtc gctccggtta gcctggagtg tcaagggcca gcagcctgaa actcctgggg gtggctctgg tgttgacaca gccgagcaag ctcctaggct gtggcagtgg ttgcagttta aggtggaaat cgtgcccagc aggacaccct acgaagaccc agacaaagcc tcctgcacca tcccagcccc tgtacaccct tggtcaaagg agaacaacta gcaagctcac tgcatgaggc ggcacaacaa tctcttcctc tggagcagag ctcattcact gatgggcaat ggtcactatc ggcctcggac ccagggcacc cggaggtgga gtctccagcc tgaaaatgtt cctcatctat ctccgggaca ttactgtcaa caaatcctcg acctgaagcc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatcca caagaccacg cgtggacaag tctgcacaac ttcttccctg ctgctactct gtgaaaaagc ggctacaata attgatcctt tccgccgaca accgccatgt ctggtcactg tccggtggcg accctgtctt tacagctact tttgcaaaaa gacttcactc catcattccg agtgagccca gcgggtgcac cggacccctg ttcaactggt cagtacaaca aatggcaagg accatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc aatacaggaa ggctcccaga ccggagagtc tgaactgggt attatggtgg agtccatcag attactgtgc tctcctctgg gcggatctgg tgtctccagg tagcctggta ccttagcaga tcaccatcag ataatccgtg aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg catacgcgtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct ctcagatggc taccaccggt tctgaagatt gcgccagatg tactacctac caccgcctac acgctcagtc gggtggaggc cgggggtggc cgaaagagcc ccaacagaaa aggtattcca cagcctagag gacattcggc caaaactcac cctcttcccc cgtggtggtg cgtggaggtg tgtggtcagc cgcggtctcc gcagccccga ccaggtcagc ggagagcaat cggctccttc cgtcttctca ctccctgtct aggtcattct

120

180

240

300

360

420

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660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

- 85 042409 ccgaattctc aggtccagct ggtggagtct gggggcggag tggtgcagcc tgggcggtca1620 ctgaggctgt cctgcaaggc ttctggctac acctttacta gatctacgat gcactgggta1680 aggcaggccc ctggacaagg tctggaatgg attggataca ttaatcctag cagtgcttat1740 actaattaca atcagaaatt caaggacagg ttcacaatca gcgcagacaa atccaagagc1800 acagccttcc tgcagatgga cagcctgagg cccgaggaca ccggcgtcta tttctgtgca1860 cggccccaag tccactatga ttacaacggg tttccttact ggggccaagg gactcccgtc1920 actgtctcta gcggtggcgg agggtctggg ggtggcggat ccggaggtgg tggctctgca1980 caagacatcc agatgaccca gtctccaagc agcctgtctg caagcgtggg ggacagggtc2040 accatgacct gcagtgccag ctcaagtgta agttacatga actggtacca gcagaagccg2100 ggcaaggccc ccaaaagatg gatttatgac tcatccaaac tggcttctgg agtccctgct2160 cgcttcagtg gcagtgggtc tgggaccgac tataccctca caatcagcag cctgcagccc2220 gaagatttcg ccacttatta ctgccagcag tggagtcgta acccacccac gttcggaggg2280 gggaccaagc tacaaattac atcctccagc taa2313 (SEQ ID NO:69)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for CAS105:

EVQLVQSGAE NRKFKGQVTI SGGGGSGGGG PGQAPRLLIY QGTKVEIKSS DVSHEDPEVK NKALPAPIEK GQPENNYKTT PGQRHNNSSL RQAPGQGLEW RPQVHYDYNG TMTCSASSSV

VKKPGESLKI SADKSISTAY SGGGGSGGGG FAKTLAEGIP SEPKSSDKTH FNWYVDGVEV TISKAKGQPR PPVLDSDGSF NTGTQMAGHS IGYINPSSAY FPYWGQGTPV SYMNWYQQKP

SCKGSGYSFT LQWSSLKASD SEIVLTQSPA ARFSGSGSGT TSRRSPAREA HNAKTKPREE EPQVYTLPPS FLYSKLTVDK PNSQVQLVES TNYNQKFKDR TVSSGGGGSG GKAPKRWIYD

GYNMNWVRQM TAMYYCARSV TLSLSPGERA DFTLTISSLE AGAPSVFLFP QYNSTYRWS RDELTKNQVS SRWQQGNVFS GGGWQPGRS FTISADKSKS GGGSGGGGSA SSKLASGVPA

PGKGLEWMGN GPFDSWGQGT TLSCRASENV PEDFAVYYCQ PKPKDTLMIS VLTVLHQDWL LTCLVKGFYP CSVMHEALHN LRLSCKASGY TAFLQMDSLR QDIQMTQSPS RFSGSGSGTD

IDPYYGGTTY

LVTVSSGGGG

YSYLAWYQQK

HHSDNPWTFG

RTPEVTCVW

NGKAYACAVS

SDIAVEWESN

HYTQKSLSLS

TFTRSTMHWV

PEDTGVYFCA

SLSASVGDRV

YTLTSSLQP

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360

420

480

540

600

660

720

EDFATYYCQQ WSRNPPTFGG GTKLQITSSS

750 (SEQ ID NO:70)

DNA sequence for anti-SP37 X TSC445:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggagagtc tctgaagatt tcctgtaagg gctccggtta ctcattcact ggctacaata tgaactgggt gcgccagatg cccgggaaag gcctggagtg gatgggcaat attgatcctt attatggtgg tactacctac aaccggaagt tcaagggcca ggtcactatc tccgccgaca agtccatcag caccgcctac ctgcaatgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acgctcagtc ggccctttcg actcctgggg ccagggcacc ctggtcactg tctcctctgg gggtggaggc tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctgg cgggggtggc tctgaaattg tgttgacaca gtctccagcc accctgtctt tgtctccagg cgaaagagcc

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540

- 86 042409 accctctcct gccgagcaag tgaaaatgtt tacagctact tagcctggta ccaacagaaa600 cctggccagg ctcctaggct cctcatctat tttgcaaaaa ccttagcaga aggtattcca660 gccaggttca gtggcagtgg ctccgggaca gacttcactc tcaccatcag cagcctagag720 cctgaagatt ttgcagttta ttactgtcaa catcattccg ataatccgtg gacattcggc780 caagggacca aggtggaaat caaatcctcg agtgagccca aatcttctga caaaactcac840 acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc900 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc1080 gtcctcaccg tcctgcacca ggactggctg aatggcaagg catacgcgtg cgcggtctcc1140 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc1260 ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc1380 ttcctctaca gcaa gctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct1500 ccgggtcaga ggcacaacaa ttcttccctg aatacaggaa ctcagatggc aggtcattct1560 ccgaattctc aggtccagct ggtgcaatct gggcctgagg tgaagaagcc tgggtcctcg1620 gtgaaggtct cctgcaaggc ttctggatat accttcagca gatctacgat gcactgggtg1680 cgacaggccc ctggacaagg gcttgagtgg ataggataca ttaatcctag cagtgcttat1740 actaattaca atcagaaatt caaggacaga gtcacgatta ccgcggacaa atccacgagc1800 acagcctaca tggagctgag cagcctgaga tctgaggaca cggccgtgta ttactgtgcg1860 agaccccaag tccactatga ttacaacggg tttccttact ggggccaagg aaccctggtc1920 accgtctcct caggtggagg cggttcaggc ggaggtggat ccggcggtgg cggatcgggt1980 ggcggcggat ctgacatcca gatgacccag tctccttcca ccctgtctgc atctgtagga2040 gacagagtca ccatgacttg cagtgccagc tcaagtgtaa gttacatgaa ctggtatcag2100 cagaaaccag ggaaagcccc taagagatgg atttatgact catccaaact ggcttctggg2160 gtcccatcaa ggttcagcgg cagtggatct gggacagagt tcactctcac catcagcagc2220 ctgcagcctg atgattttgc aacttattac tg ccaacagt ggagtcgtaa cccacccact2280 ttcggcggag ggaccaaggt ggagatcaaa cggtcctcca gctaa2325 (SEQ ID NO:71)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CB37 X TSC445:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS480

PGQRHNNSSL NTGTQMAGHS PNSQVQLVQS GPEVKKPGSS VKVSCKASGY TFSRSTMHWV540

RQAPGQGLEW IGYINPSSAY TNYNQKFKDR VTITADKSTS TAYMELSSLR SEDTAVYYCA600

RPQVHYDYNG FPYWGQGTLV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASVG660

- 87 042409

DRVTMTCSAS SSVSYMNWYQ QKPGKAPKRW IYDSSKLASG VPSRFSGSGS GTEFTLTISS

720

LQPDDFATYY CQQWSRNPPT FGGGTKVEIK RSSS

754 (SEQ ID NO:72)

DNA sequence for anti-SP37 X TSC452:

atggaagcac gaggtgcagc tcctgtaagg cccgggaaag aaccggaagt ctgcaatgga ggccctttcg tctggtggcg tctgaaattg accctctcct cctggccagg gccaggttca cctgaagatt caagggacca acatgcccac ccaaaaccca gacgtgagcc cataatgcca gtcctcaccg aacaaagccc gaaccacagg ctgacctgcc gggcagccgg ttcctctaca tgctccgtga ccgggtcaga ccgaattctc ctgaggctgt aggcaggccc actaattaca acagccttcc cggccccaag actgtctcta ggcggcggat gacagagtca cagaaaccag gtcccatcaa ctgcagcctg ttcggcggag cagcgcagct tggtgcagtc gctccggtta gcctggagtg tcaagggcca gcagcctgaa actcctgggg gtggctctgg tgttgacaca gccgagcaag ctcctaggct gtggcagtgg ttgcagttta aggtggaaat cgtgcccagc aggacaccct acgaagaccc agacaaagcc tcctgcacca tcccagcccc tgtacaccct tggtcaaagg agaacaacta gcaagctcac tgcatgaggc ggcacaacaa aggtccagct cctgcaaggc ctggacaagg atcagaaatt tgcagatgga tccactatga gcggtggcgg ctgacatcca ccatgacttg ggaaagcccc ggttcagcgg atgattttgc ggaccaaggt tctcttcctc tggagcagag ctcattcact gatgggcaat ggtcactatc ggcctcggac ccagggcacc cggaggtgga gtctccagcc tgaaaatgtt cctcatctat ctccgggaca ttactgtcaa caaatcctcg acctgaagcc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatcca caagaccacg cgtggacaag tctgcacaac ttcttccctg ggtggagtct ttctggctac tctggaatgg caaggacagg cagcctgagg ttacaacggg agggtctggg gatgacccag cagtgccagc taagagatgg cagtggatct aacttattac ggagatcaaa ctgctactct gtgaaaaagc ggctacaata attgatcctt tccgccgaca accgccatgt ctggtcactg tccggtggcg accctgtctt tacagctact tttgcaaaaa gacttcactc catcattccg agtgagccca gcgggtgcac cggacccctg ttcaactggt cagtacaaca aatggcaagg accatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc aatacaggaa gggggcggag acctttacta attggataca ttcacaatca cccgaggaca tttccttact ggtggcggat tctccttcca tcaagtgtaa atttatgact gggacagagt tgccaacagt cggtcctcca ggctcccaga ccggagagtc tgaactgggt attatggtgg agtccatcag attactgtgc tctcctctgg gcggatctgg tgtctccagg tagcctggta ccttagcaga tcaccatcag ataatccgtg aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg catacgcgtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct ctcagatgg c tggtgcagcc gatctacgat ttaatcctag gcgcagacaa ccggcgtcta ggggccaagg ccggcggtgg ccctgtctgc gttacatgaa catccaaact tcactctcac ggagtcgtaa gctaa taccaccggt tctgaagatt gcgccagatg tactacctac caccgcctac acgctcagtc gggtggaggc cgggggtggc cgaaagagcc ccaacagaaa aggtattcca cagcctagag gacattcggc caaaactcac cctcttcccc cgtggtggtg cgtggaggtg tgtggtcagc cgcggtctcc gcagccccga ccaggtcagc ggagagcaat cggctccttc cgtcttctca ctccctgtct aggtcattct tgggcggtca gcactgggta cagtgcttat atccaagagc tttctgtgca gactcccgtc cggatcgggt atctgtagga ctggtatcag ggcttctggg catcagcagc ccccaccact

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2325

- 88 042409 (SEQ ID NO:73)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CB37 X TSC452:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS480

PGQRHNNSSL NTGTQMAGHS PNSQVQLVES GGGWQPGRS LRLSCKASGY TFTRSTMHWV540

RQAPGQGLEW IGYINPSSAY TNYNQKFKDR FTISADKSKS TAFLQMDSLR PEDTGVYFCA600

RPQVHYDYNG FPYWGQGTPV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASVG660

DRVTMTCSAS SSVSYMNWYQ QKPGKAPKRW IYDSSKLASG VPSRFSGSGS GTEFTLTISS720

LQPDDFATYY CQQWSRNPPT FGGGTKVEIK RSSS754 (SEQ ID NO:74)

DNA sequence for anti-SP37 X TSC453:

atggaagcac gaggtgcagc tcctgtaagg cccgggaaag aaccggaagt ctgcaatgga ggccctttcg tctggtggcg tctgaaattg accctctcct cctggccagg gccaggttca cctgaagatt caagggacca acatgcccac ccaaaaccca gacgtgagcc cataatgcca gtcctcaccg aacaaagccc gaaccacagg ctgacctgcc gggcagccgg cagcgcagct tggtgcagtc gctccggtta gcctggagtg tcaagggcca gcagcctgaa actcctgggg gtggctctgg tgttgacaca gccgagcaag ctcctaggct gtggcagtgg ttgcagttta aggtggaaat cgtgcccagc aggacaccct acgaagaccc agacaaagcc tcctgcacca tcccagcccc tgtacaccct tggtcaaagg agaacaacta tctcttcctc tggagcagag ctcattcact gatgggcaat ggtcactatc ggcctcggac ccagggcacc cggaggtgga gtctccagcc tgaaaatgtt cctcatctat ctccgggaca ttactgtcaa caaatcctcg acctgaagcc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatcca caagaccacg ctgctactct gtgaaaaagc ggctacaata attgatcctt tccgccgaca accgccatgt ctggtcactg tccggtggcg accctgtctt tacagctact tttgcaaaaa gacttcactc catcattccg agtgagccca gcgggtgcac cggacccctg ttcaactggt cagtacaaca aatggcaagg accatctcca cgggatgagc agcgacatcg cctcccgtgc ggctcccaga ccggagagtc tgaactgggt attatggtgg agtccatcag attactgtgc tctcctctgg gcggatctgg tgtctccagg tagcctggta ccttagcaga tcaccatcag ataatccgtg aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg catacgcgtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga taccaccggt tctgaagatt gcgccagatg tactacctac caccgcctac acgctcagtc gggtggaggc cgggggtggc cgaaagagcc ccaacagaaa aggtattcca cagcctagag gacattcggc caaaactcac cctcttcccc cgtggtggtg cgtggaggtg tgtggtcagc cgcggtctcc gcagccccga ccaggtcagc ggagagcaat cggctccttc

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- 89 042409

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500 ccgggtcaga ggcacaacaa ttcttccctg aatacaggaa ctcagatggc aggtcattct 1560 ccgaattctc aggtccagct ggtgcaatct gggcctgagg tgaagaagcc tgggtcctcg 1620 gtgaaggtct cctgcaaggc ttctggatat accttcagca gatctacgat gcactgggtg 1680 cgacaggccc ctggacaagg gcttgagtgg ataggataca ttaatcctag cagtgcttat 1740 actattaca atcagaaatt caaggacaga gtcacgatta ccgcggacaa atccacgagc 1800 acagcctaca tggagctgag cagcctgaga tctgaggaca cggccgtgta ttactgtgcg 1860 agaccccaag tccactatga ttacaacggg ttttccttact ggggccaagg aaccctggtc 1920 accgtctcct caggtgggagg cggttcaggc ggaggtggat ccgggaggtgg tggctctggt 1980 ggcggcggat ctgacatcca gatgacccag tctccaagca gcctgtctgc aagcgtgggg 2040 gacagggtca ccatgacctg cagtgccagc tcaagtgtaa gttacatgaa ctggtaccag 2100 cagaagccgg gcaaggcccc caaaagatgg attatgact catccaaact ggcttctgga 2160 gtccctgctc gcttcagtgg cagtgggtct gggaccgact ataccctcac aatcagcagc 2220 ctgcagcccg aagatttcgc cacttattac tgccagcagt ggagtcgtaa cccacccg 2280 ttcggagggg ggaccaagct acaattaca tcctccagct aa 2322

(SEQ ID NO:75)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CB37 X TSC453:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS480

PGQRHNNSSL NTGTQMAGHS PNSQVQLVQS GPEVKKPGSS VKVSCKASGY TFSRSTMHWV540

RQAPGQGLEW IGYINPSSAY TNYNQKFKDR VTITADKSTS TAYMELSSLR SEDTAVYYCA600

RPQVHYDYNG FPYWGQGTLV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG660

DRVTMTCSAS SSVSYMNWYQ QKPGKAPKRW IYDSSKLASG VPARFSGSGS GTDYTLTISS720

LQPEDFATYY CQQWSRNPPT FGGGTKLQIT SSS753 (SEQ ID NO:76)

DNA sequence for anti-SP37 X TSC454:

atggaagcac cagcgcagct tctcttcctc gaggtgcagc tggtgcagtc tggagcagag tcctgtaagg gctccggtta ctcattcact cccgggaaag gcctggagtg gatgggcaat aaccggaagt tcaagggcca ggtcactatc ctgcaatgga gcagcctgaa ggcctcggac ctgctactct ggctcccaga taccaccggt gtgaaaaagc ccggagagtc tctgaagatt ggctacaata tgaactgggt gcgccagatg attgatcctt attatggtgg tactacctac tccgccgaca agtccatcag caccgcctac accgccatgt attactgtgc acgctcagtc

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- 90 042409 ggccctttcg actcctgggg ccagggcacc ctggtcactg tctcctctgg gggtggaggc420 tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctgg cgggggtggc480 tctgaaattg tgttgacaca gtctccagcc accctgtctt tgtctccagg cgaaagagcc540 accctctcct gccgagcaag tgaaaatgtt tacagctact tagcctggta ccaacagaaa600 cctggccagg ctcctaggct cctcatctat tttgcaaaaa ccttagcaga aggtattcca660 gccaggttca gtggcagtgg ctccgggaca gacttcactc tcaccatcag cagcctagag720 cctgaagatt ttgcagttta ttactgtcaa catcattccg ataatccgtg gacattcggc780 caagggacca aggtggaaat caaatcctcg agtgagccca aatcttctga caaaactcac840 acatgcccac cgtgcccagc acctgaagcc gcgggtgcac cgtcagtctt cctcttcccc900 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc1080 gtcctcaccg tcctgcacca ggactggctg aatggcaagg catacgcgtg cgcggtctcc1140 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga1200 gaaccacagg tgtacac cct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc1260 ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct1500 ccgggtcaga ggcacaacaa ttcttccctg aatacaggaa ctcagatggc aggtcattct1560 ccgaattctc aggtccagct ggtgcaatct gggcctgagg tgaagaagcc tgggtcctcg1620 gtgaaggtct cctgcaaggc ttctggatat accttcagca gatctacgat gcactgggtg1680 cgacaggccc ctggacaagg gcttgagtgg ataggataca ttaatcctag cagtgcttat1740 actaattaca atcagaaatt caaggacaga gtcacgatta ccgcggacaa atccacgagc1800 acagcctaca tggagctgag cagcctgaga tctgaggaca cggccgtgta ttactgtgcg1860 agaccccaag tccactatga ttacaacggg tttccttact ggggccaagg aaccctggtc1920 accgtctcct caggtggagg cggttcaggc ggaggtggat ccggcggtgg cggatcgggt1980 ggcggcggat ctgacatcca gatgacccag tctccttcca ccctgtctgc atctgtagga2040 gacagagtca ccatgacttg cagtgccagc tcaag tgtaa gttacatgaa ctggtatcag2100 cagaaaccag ggaaagcccc taagagatgg atttatgact catccaaact ggcttctggg2160 gtcccatcaa ggttcagcgg cagtggatct gggacagatt tcactctcac catcagcagc2220 ctgcagcctg atgattttgc aacttattac tgccaacagt ggagtcgtaa cccacccact2280 ttcggcggag ggaccaaggt ggagatcaaa cggtcctcca gctaa2325 (SEQ ID NO:77)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CB37 X TSC454:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

- 91 042409

GQPENNYKTT

PGQRHNNSL

RQAPGQGLEW

RPQVHYDYNG

DRVTMTCSAS

PPVLDSDGSF

NTGTQMAGHS

IGYINPSSAY

FPYWGQGTLV

SSVSYMNWYQ

FLYSKLTVDK

PNSQVQLVQS

TNYNQKFKDR

TVSSGGGGSG

QKPGKAPKRW

SRWQQGNVFS

GPEVKKPGSS

VTITADKSTS

GGGSGGGGSG

IYDSSKLASG

CSVMHEALHN

VKVSCKASGY

TAYMELSSLR

GGGSDIQMTQ

VPSRFSGSGS

HYTQKSLSLS

TFSRSTMHWV

SEDTAVYYCA

SPSTLSASVG

GTDFTLTISS

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LQPDDFATYY CQQWSRNPPT FGGGTKVEIK RSSS

754 (SEQ ID NO:78)

DNA sequence for anti-SP37 X TSC455:

atggaagcac gaggtgcagc tcctgtaagg cccgggaaag aaccggaagt ctgcaatgga ggccctttcg tctggtggcg tctgaaattg accctctcct cctggccagg gccaggttca cctgaagatt caagggacca acatgcccac ccaaaaccca gacgtgagcc cataatgcca gtcctcaccg aacaaagccc gaaccacagg ctgacctgcc gggcagccgg ttcctctaca tgctccgtga ccgggtcaga ccgaattctc gtgaaggtct cgacaggccc actaattaca acagcctaca agaccccaag accgtctcct ggcggcggat gacagagtca cagaaaccag cagcgcagct tggtgcagtc gctccggtta gcctggagtg tcaagggcca gcagcctgaa actcctgggg gtggctctgg tgttgacaca gccgagcaag ctcctaggct gtggcagtgg ttgcagttta aggtggaaat cgtgcccagc aggacaccct acgaagaccc agacaaagcc tcctgcacca tcccagcccc tgtacaccct tggtcaaagg agaacaacta gcaagctcac tgcatgaggc ggcacaacaa aggtccagct cctgcaaggc ctggacaagg atcagaaatt tggagctgag tccactatga caggtggagg ctgacatcca ccatgacttg ggaaagcccc tctcttcctc tggagcagag ctcattcact gatgggcaat ggtcactatc ggcctcggac ccagggcacc cggaggtgga gtctccagcc tgaaaatgtt cctcatctat ctccgggaca ttactgtcaa caaatcctcg acctgaagcc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatcca caagaccacg cgtggacaag tctgcacaac ttcttccctg ggtgcaatct ttctggatat gcttgagtgg caaggacaga cagcctgaga ttacaacggg cggttcaggc gatgacccag cagtgccagc taagagatgg ctgctactct gtgaaaaagc ggctacaata attgatcctt tccgccgaca accgccatgt ctggtcactg tccggtggcg accctgtctt tacagctact tttgcaaaaa gacttcactc catcattccg agtgagccca gcgggtgcac cggacccctg ttcaactggt cagtacaaca aatggcaagg accatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc aatacaggaa gggcctgagg accttcagca ataggataca gtcacgatta tctgaggaca tttccttact ggaggtggat tctccttcca tcaagtgtaa atttatgact ggctcccaga ccggagagtc tgaactgggt attatggtgg agtccatcag attactgtgc tctcctctgg gcggatctgg tgtctccagg tagcctggta ccttagcaga tcaccatcag ataatccgtg aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg catacgcgtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct ctcagatggc tgaagaagcc gatctacgat ttaatcctag ccgcggacaa cggccgtgta ggggccaagg ccggcggtgg ccctgtctgc gttacatgaa catccaaact taccaccggt tctgaagat t gcgccagatg tactacctac caccgcctac acgctcagtc gggtggaggc cgggggtggc cgaaagagcc ccaacagaaa aggtattcca cagcctagag gacattcggc caaaactcac cctcttcccc cgtggtggtg cgtggaggtg tgtggtcagc cgcggtctcc gcagccccga ccaggtcagc ggagagcaat cggctccttc cgtcttctca ctccctgtct aggtcattct tgggtcctcg gcactgggtg cagtgcttat atccacgagc ttactgtgcg aaccctggtc cggatcgggt atctgtagga ctggtatcag ggcttctggg

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gtcccatcaa ggttcagcgg cagtggatct gggacagagt atactctcac catcagcagc 2220 ctgcagcctg atgattttgc aacttattac tgccaacagt ggagtcgtaa ccccccact 2280 ttcggcggag ggaccaaggt ggagatcaaa cggtcctcca gctaa 2325

(SEQ ID NO:79)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CB37 X TSC455:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS480

PGQRHNNSSL NTGTQMAGHS PNSQVQLVQS GPEVKKPGSS VKVSCKASGY TFSRSTMHWV540

RQAPGQGLEW IGYINPSSAY TNYNQKFKDR VTITADKSTS TAYMELSSLR SEDTAVYYCA600

RPQVHYDYNG FPYWGQGTLV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASVG660

DRVTMTCSAS SSVSYMNWYQ QKPGKAPKRW IYDSSKLASG VPSRFSGSGS GTEYTLTISS720

LQPDDFATYY CQQWSRNPPT FGGGTKVEIK RSSS754 (SEQ ID NO:80)

DNA sequence for anti-CO37 X TSC456:

atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt60 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggagagtc tctgaagatt120 tcctgtaagg gctccggtta ctcattcact ggctacaata tgaactgggt gcgccagatg180 cccgggaaag gcctggagtg gatgggcaat attgatcctt attatggtgg tactacctac240 aaccggaagt tcaagggcca ggtcactatc tccgccgaca agtccatcag caccgcctac300 ctgcaatgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acgctcagtc360 ggccctttcg actcctgggg ccagggcacc ctggtcactg tctcctctgg gggtggaggc420 tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctgg cgggggtggc480 tctgaaattg tgttgacaca gtctccagcc accctgtctt tgtctccagg cgaaagagcc540 accctctcct gccgagcaag tgaaaatgtt tacagctact tagcctggta ccaacagaaa600 cctggccagg ctcctaggct cctcatctat tttgcaaaaa ccttagcaga aggtattcca660 gccaggttca gtggcagtgg ctccgggaca gacttcactc tcaccatcag cagcctagag720 cctgaagatt ttgcagttta ttactgtcaa catcattccg ataatccgtg gacattcggc780 caagggacca aggtggaaat caaatcctcg agtgagccca aatcttctga caaaactcac840 acatgcccac cgtgcccagc acctgaagcc gc gggtgcac cgtcagtctt cctcttcccc900 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc1080 gtcctcaccg tcctgcacca ggactggctg aatggcaagg catacgcgtg cgcggtctcc1140 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc1260

- 93 042409 ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg ggagagcaat1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct1500 ccgggtcaga ggcacaacaa ttcttccctg aatacaggaa ctcagatggc aggtcattct1560 ccgaattctc aggtccagct ggtgcaatct gggcctgagg tgaagaagcc tgggtcctcg1620 gtgaaggtct cctgcaaggc ttctggatat accttcagca gatctacgat gcactgggtg1680 cgacaggccc ctggacaagg gcttgagtgg ataggataca ttaatcctag cagtgcttat1740 actaattaca atcagaaatt caaggacaga gtcacgatta ccgcggacaa atccacgagc1800 acagcctaca tggagctgag cagcctgaga tctgaggaca cggccgtgta ttactgtgcg1860 agaccccaag tccactatga ttacaacggg tttccttact ggggccaagg aaccctggtc1920 accgtctcct caggtggagg cggttcaggc ggaggtggat ccggcggtgg cggatcgggt1980 ggcggcggat ctgacatcca gatgacccag tctccttcca ccctgtctgc atctgtagga2040 gacagagtca ccatgacttg cagtgccagc tcaagtgtaa gttacatgaa ctggtatcag2100 cagaaacc ag ggaaagcccc taagagatgg atttatgact catccaaact ggcttctggg2160 gtcccatcaa ggttcagcgg cagtggatct gggacagatt atactctcac catcagcagc2220 ctgcagcctg atgattttgc aacttattac tgccaacagt ggagtcgtaa cccacccact2280 ttcggcggag ggaccaaggt ggagatcaaa cggtcctcca gctaa2325 (SEQ ID NO:81)

The signal sequence is represented by residues from 1 to 60

Mature protein sequence for anti-CO37 X TSC456:

EVQLVQSGAE VKKPGESLKI SCKGSGYSFT GYNMNWVRQM PGKGLEWMGN IDPYYGGTTY60

NRKFKGQVTI SADKSISTAY LQWSSLKASD TAMYYCARSV GPFDSWGQGT LVTVSSGGGG120

SGGGGSGGGG SGGGGSGGGG SEIVLTQSPA TLSLSPGERA TLSCRASENV YSYLAWYQQK180

PGQAPRLLIY FAKTLAEGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ HHSDNPWTFG240

QGTKVEIKSS SEPKSSDKTH TSRRSPAREA AGAPSVFLFP PKPKDTLMIS RTPEVTCVW300

DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKAYACAVS360

NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN420

GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS480

PGQRHNNSSL NTGTQMAGHS PNSQVQLVQS GPEVKKPGSS VKVSCKASGY TFSRSTMHWV540

RQAPGQGLEW IGYINPSSAY TNYNQKFKDR VTITADKSTS TAYMELSSLR SEDTAVYYCA600

RPQVHYDYNG FPYWGQGTLV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASVG660

DRVTMTCSAS SSVSYMNWYQ QKPGKAPKRW IYDSSKLASG VPSRFSGSGS GTDYTLTISS720

LQPDDFATYY CQQWSRNPPT FGGGTKVEIK RSSS754 (SEQ ID NO:82)

Protein sequence for TSC455 anti-POPs scFv

QVQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVTITADK STSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQS PSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTEYTLTISSLQPDD FAT YYCQQWSRNPPT FGGGT KVEIKRS S S

- 94 042409 (SEQ ID NO:83)

Protein sequence for TSC456 anti-POPs scFv

QVQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVTITADK STSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQS PSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDD FAT YYCQQWSRNPPT FGGGT KVEIKRS S S (SEQ ID NO:84)

Protein sequence for DRA222 anti-SBZ scFv

QVQLVESGGGWQPGRSLRLSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRFTISADK SKSTAFLQMDSLRPEDTGVYFCARPQVHYDYNGFPYWGQGTPVTVSSGGGGSGGGGSGGGGSAQDIQMTQSPSS LSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPARFSGSGSGTDYTLTISSLQPEDFAT YYCQQWSRNPPTFGGGTKLQITSSS (SEQ ID NO:85)

Protein sequence for the variable heavy domain of TSC455 and TSC456

QVQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVTITADK STSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSS (SEQ ID NO:86)

Protein sequence for the variable heavy domain of DRA222

QVQLVESGGGWQPGRSLRLSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRFTISADK SKSTAFLQMDSLRPEDTGVYFCARPQVHYDYNGFPYWGQGTPVTVSS (SEQ ID NO:87)

Protein sequence for the TSC455 variable light domain

DIQMTQSPSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTEYTLTI SSLQPDDFATYYCQQWSRNPPTFGGGTKVEIKRS (SEQ ID NO:88)

Protein sequence for the TSC456 variable light domain

- 95 042409

DIQMTQSPSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDYTLTI SSLQPDDFATYYCQQWSRNPPTFGGGTKVEIKRS (SEQ ID NO:89)

Protein sequence for the DRA222 variable light domain

DIQMTQSPSSLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPARFSGSGSGTDYTLTI SSLQPEDFATYYCQQWSRNPPTFGGGTKLQITS (SEQ ID NO:90)

Cris7 and DRA222 VH CDR1 (Kabat) RSTMH (SEQ ID NO:91)

Cris7 and DRA222 VH CDR2 (Kabat) YINPSSAYTNYNQKFK (SEQ ID NO:92)

Cris7 and DRA222 VH CDR3 (Kabat) QVHYDYNGFPY (SEQ ID NO:93)

Cris7 and DRA222 VL CDR1 (Kabat) SASSSVSYMN (SEQ ID NO:94)

Cris7 and DRA222 VL CDR2 (Kabat) DSSKLAS (SEQ ID NO:95)

Cris7 and DRA222 VL CDR3 (Kabat) QQWSRNPPT (SEQ ID NO:96)

Cris7 and DRA222 VH CDR1 (IMGT) GYTFTRST (SEQ ID NO: 199)

Cris7 and DRA222 VH CDR2 (IMGT) INPSSAYT (SEQ ID N0:200)

Cris7 and DRA222 VH CDR3 (IMGT) ARPQVHYDYNGFPY (SEQ ID NO:201)

Cris7 and DRA222 VL CDR1 (IMGT) ASSSVSY (SEQ ID NO:202)

Cris7 and DRA222 VL CDR2 (IMGT) DSS (SEQ ID NO:203)

Cris7 and DRA222 VL CDR3 (IMGT) QQWSRNPPT (SEQ ID NO:204)

DNA sequence for ROR133:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca

- 96 042409 aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaac gggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacatcc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO:97)

Mature protein sequence for ROR133:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypiswvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyygdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytf trstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO:98)

DNA sequence for ROR193:

- 97 042409 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccct gaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatc gggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagagtatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO:99)

Mature protein sequence for ROR193:

Diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypiswvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyygdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgy

- 98 042409 tfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqg tlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvp srfsgsgsgteytltisslqpddfatyycqqwsmpptfgggtkveikrsss (SEQ ID NO: 100)

DNA sequence for ROR134:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gccgactcggtgaagggccggttcaccatctcccggcactcctccaagaacaccctgtacctgcagatgaactccctgcgggccgaggac accgccgtgtacttctgcgcccggggctactccacctactacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacat cc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO: 101)

- 99 042409

Mature protein sequence for ROR134:

Diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpggslrlsctasgsdindypiswvrqapgkglewi gfinsggstwyadsvkgrftisrhsskntlylqmnslraedtavyfcargystyygdfniwgqgtlvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytft rstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO: 102)

DNA sequence for ROR189:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gccgactcggtgaagggccggttcaccatctcccggcactcctccaagaacaccctgtacctgcagatgaactccctgcgggccgaggac accgccgtgtacttctgcgcccggggctactccacctactacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat

- 100 042409 acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO: 103)

Mature protein sequence for ROR189:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpggslrlsctasgsdindypiswvrqapgkglewi gfinsggstwyadsvkgrftisrhsskntlylqmnslraedtavyfcargystyygdfniwgqgtlvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgyt fsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqgt Ivtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvps rfsgsgsgtdytltisslqpddfatyycqqwsmpptfgggtkveikrsss (SEQ ID NO: 104)

DNA sequence for ROR154:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg

- 101 042409 tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacatcc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctca agtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO: 105)

Mature protein sequence for ROR154:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytf trstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO: 106)

DNA sequence for ROR185:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct

- 102 042409 gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaa cgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO: 107)

Mature protein sequence for ROR185:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgy tfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqg tlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvp srfsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrsss (SEQ ID NO: 108)

- 103 042409

DNA sequence for ROR179:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctactggacctccttcgg cggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatctg aggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatcaac gactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtacgc ctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggacac cgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcgagt gagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc a actggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgt cctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacat cc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO: 109)

Mature protein sequence for ROR179:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsywtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytf trstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO: 110)

- 104 042409

DNA sequence for ROR186:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctactggacctccttcgg cggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatctg aggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatcaac gactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtacgc ctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggacac cgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcgagt gagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc a actggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgt cctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgg a tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO:111)

Mature protein sequence for ROR186:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsywtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgkglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly

- 105 042409 skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgy tfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqg tlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvp srfsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrsss (SEQ ID NO: 112)

DNA sequence for ROR181:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcaccgtgtccggcaccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gccgactcggtgaagggccggttcaccatctcccggcactcctccaagaacaccctgtacctgcagatgaactccctgcgggccgaggac accgccgtgtacttctgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacat cc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO: 113)

- 106 042409

Mature protein sequence for ROR181:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpggslrlsctvsgtdindypiswvrqapgkglewi gfinsggstwyadsvkgrftisrhsskntlylqmnslraedtavyfcargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytft rstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytlti s si qpedfatyy c qqwsmpptfgggtkl qi ts ss (SEQ ID NO: 114)

DNA sequence for ROR191:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcaccgtgtccggcaccgacatca acgactaccccatctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gccgactcggtgaagggccggttcaccatctcccggcactcctccaagaacaccctgtacctgcagatgaactccctgcgggccgaggac accgccgtgtacttctgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg

- 107 042409 ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO: 115)

Mature protein sequence for ROR191:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgkppklliyrasnlasgvpsrfsgsgsgtdftltisslqpedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpggslrlsctvsgtdindypiswvrqapgkglewi gfinsggstwyadsvkgrftisrhsskntlylqmnslraedtavyfcargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgyt fsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqgt Ivtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvps rfsgsgsgtdytltisslqpddfatyycqqwsmpptfgggtkveikrsss (SEQ ID NO: 116)

DNA sequence for ROR182:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc cagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggctccggctccggcaccgacttc accctgaccatctcctccctggaggccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatc tgaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggccagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa

- 108 042409 ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacatcc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtc cctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagctaa (SEQ ID NO: 117)

Mature protein sequence for ROR182:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgqppklliyrasnlasgvpdrfsgsgsgtdftltissleaedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgqglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvesgggvvqpgrslrlsckasgytf trstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO: 118)

DNA sequence for ROR192:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc cagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggctccggctccggcaccgacttc accctgaccatctcctccctggaggccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatc

- 109 042409 tgaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggccagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagctaa (SEQ ID NO: 119)

Mature protein sequence for ROR192:

diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgqppklliyrasnlasgvpdrfsgsgsgtdftltissleaedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgqglewi gfinsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckasgy tfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqg tlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvp srfsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrsss (SEQ ID NO: 120)

- 110 042409

Химерное биспецифическое анти-СП123 (VLVH) х анти-СПЗ (TSC456) scFv-Fc-scFv Dimmsqspsslavsvgekftmtckssqslffgstqknylawyqqkpgqspklliywastresgvpdrftgsgsgtdftlaissvmped lavyycqqyynypwtfgggtkleikggggsggggsggggsggggsvqlqesgpglvkpsqslsltcsvtdysitsgyywnwirqfp gnklewmgyisydgsnnynpslknrisitrdtsknqfflklssvttedtatyycsrgegfyfdswgqgttltvsssepkssdkthtcppcp apeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng keykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsff lyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvsckas gytfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywg qgtlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasg vpsrfsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrsss (SEQ ID NO: 197)

Химерное биспецифическое анти-СП123 (VHVL) x анти-СОЗ (TSC456) scFv-Fc-scFv Vqlqesgpglvkpsqslsltcsvtdysitsgyywnwirqfpgnklewmgyisydgsnnynpslknrisitrdtsknqfflklssvttedt atyycsrgegfyfdswgqgttltvssggggsggggsggggsggggsdimmsqspsslavsvgekftmtckssqslffgstqknylaw yqqkpgqspklliywastresgvpdrftgsgsgtdftlaissvmpedlavyycqqyynypwtfgggtkleiksssepkssdkthtcppc papeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwln gkeykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgs fflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspgsqvqlvqsgpevkkpgssvkvscka sgytfsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpyw gqgtlvtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklas gvpsrfsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrsss (SEQ ID NO: 198)

DNA sequence for TSC471:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgatatccagatgacccagtctccatccgccatg tctgcatctgtaggagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcagaaaccagggaa agttcctaagctccgcatccattctggatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctca ccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtggtggctctcaggtccagctggtacagtctggggctgagg tgaagaagcctggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactactacatgcactgggtgcgacaggcccct ggacaagggcttgagtggatgggatattttaatccttataatgattatactagatacgcacagaagttccagggcagagtcaccatgaccagg gacacgtctatcagcacagcctacatggagctgagcagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttacta cgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg gaggtgcataatgccaaga caaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga

- 111 042409 atacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaca gcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccctgtctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggctctccttcacaggtgcag ctggtgcagtctggtcctgaggtgaaaaagcctggctccagcgtgaaggtgtcctgcaaggccagcggatacacctttagccggtccacca tgcattgggtgaggcaggctcctggacagggcctggagtggatcggctacatcaaccccagcagcgcttataccaactacaatcagaagtt taaggaccgggtgaccatcaccgccgataagtccaccagcaccgcctacatggagctgtccagcctgaggagcgaggataccgccgtgt actattgcgcccggccccaggtccattacgactacaacggcttcccctattggggccagggaaccctggtgaccgtgtccagcggtggcg gtggcagcggcggcggcggctctggcggaggtggcagcggcggagggggctccgacattcagatgacccagtccccctccaccctgt ccgctagcgtgggcgatcgggtgaccatgacctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaagc ccggcaag gctcccaagaggtggatttacgactccagcaagctggcctctggtgtccccagcaggttctctggtagcggcagcggcacagactacacc ctgaccatctcctccctgcagcccgacgatttcgccacctactattgccagcagtggtcccggaatccccctacctttggcggcggcaccaa ggtggagatcaagaggagctaa (SEQ: 205 ID)

Mature protein sequence for TSC471:

Diqmtqspsamsasvgdrvtitcrasksiskylawfqqkpgkvpklrihsgstlqsgvpsrfsgsgsgteftltisslqpedfatyycqqh ieypwtfgqgtkveikrggggsggggsggggsqvqlvqsgaevkkpgasvkvsckasgytftdyymhwvrqapgqglewmgyf npyndytryaqkfqgrvtmtrdtsistaymelsslrsddtavyycarsdgyydamdywgqgttvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspsqvqlvqsgpevkkpgssvkvsckasgytf srstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqgtl vtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvpsr fsgsgsgtdytltisslqpddfatyycqqwsrnpptfgggtkveikrs (SEQ ID NO:206)

DNA sequence for ROR243:

atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgacaccacaggcgacatccagatgacccagtccccttcctccct gtccgctagcgtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactccaatctggcctggttccagcagaagcccgg acagccccccaagctgctgatttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatccggcagcggcaccgacttc accctgaccatctcctccctggaggccgaggatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcggacctccttcg gcggcggcaccaaggtggagatcaagggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggc agcgaggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtccctgaggctgtcctgcacagccagcggctccgac atcaacgactaccccatcacctgggtgaggcaggctcctggccagggcctggaatggatcggctttatcaacagcggcggcagcacctg

- 112 042409 gtatgcttcctgggtgaagggccggttcaccattagcagggacgactccaagtccattgcctacctgcagatgaactccctgaagaccgag gacaccgccgtgtactactgcgccaggggctacagcacctattaccgggactttaacatctggggccagggcacactggtcaccgtgtcct cgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcga gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgt cttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggagggggttcag gtgggggaggttctggcggcgggggaagcccttcacaggtgcaactggtgcagagtggacccgaggttaaaaaaccag ggtcctccgtt aaggttagctgcaaagcctctggctacacattttccaggagtacaatgcactgggtgaggcaggctcctggacagggactcgagtggatcg ggtatatcaacccatctagcgcctataccaattacaaccaaaagtttaaggaccgagttaccattaccgctgacaaatccaccagtacagctta tatggagctgtcatctcttaggtccgaggacactgctgtttattactgcgctcgtcctcaggttcactatgactataatggttttccctactggggt cagggaaccctggtgactgtctcttctggcggtggaggcagcggtgggggtgggtctggaggcggtggcagtggcggcggaggctctg atattcagatgactcagtctcctagcactctcagcgccagcgtgggggatcgtgtgacaatgacttgctccgctagcagtagtgtgtcttacat gaattggtatcagcagaagcccgggaaagcacctaagcgctggatctatgactcttccaagctggcaagtggtgtcccctcacggttctctg gctcaggttctggtactgactatactttgactatctcctccctccagcccgatgatttcgctacctattattgtcagcagtggagccgtaacccac ccactttcggaggcggtaccaaagtggagatcaagaggtcatga (SEQ ID NO:207)

Mature protein sequence for ROR243:

Diqmtqspsslsasvgdrvtincqasqsidsnlawfqqkpgqppklliyrasnlasgvpdrfsgsgsgtdftltissleaedvatyyclgg vgavsyrtsfgggtkveikggggsggggsggggsggggsevqlvesggglvqpgrslrlsctasgsdindypitwvrqapgqglewi gfmsggstwyaswvkgrftisrddsksiaylqmnslktedtavyycargystyyrdfniwgqgtlvtvsssepkssdkthtcppcpap eaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykcavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsffly skltvdksrwqqgnvfscsvmhealhnhytqkslslspgsggggsggggsggggspsqvqlvqsgpevkkpgssvkvsckasgyt fsrstmhwvrqapgqglewigyinpssaytnynqkfkdrvtitadkststaymelsslrsedtavyycarpqvhydyngfpywgqgt Ivtvssggggsggggsggggsggggsdiqmtqspstlsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvps rfsgsgsgtdytltisslqpddfatyycqqwsmpptfgggtkveikrs (SEQ ID NO:208)

DNA sequence for TSC266:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgatatccagatgacccagtctccatccgccatg tctgcatctgtaggagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcagaaaccagggaa agttcctaagctccgcatccattctggatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctca

- 113 042409 ccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtggtggctctcaggtccagctggtacagtctggggctgagg tgaagaagcctggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactactacatgcactgggtgcgacaggcccct ggacaagggcttgagtggatgggatattttaatccttataatgattatactagatacgcacagaagttccagggcagagtcaccatgaccagg gacacgtctatcagcacagcctacatggagctgagcagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttacta cgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaaga caaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggc gtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggc ttctatccaagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaca gcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccctgtctccgggtcagaggcacaacaattcttccctgaatacaggaactcagatggcaggtcattctccgaattctcagg tccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactagatct acgatgcactgggtaaggcaggcccctggacaaggtctggaatggattggatacattaatcctagcagtgcttatactaattacaatcagaaa ttcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcctgcagatggacagcctgaggcccgaggacaccggcgt ctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactggggccaagggactcccgtcactgtctctagcggtggcgg agggtctgggggtggcggatccggaggtggtggctctgcacaagacatccagatgacccagtctccaagcagcctgtctgcaagcgtgg gggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaaga tggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagc ctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccac gttcggaggggggaccaagctacaaattacat cctccagctaa (SEQ ID NO:209)

Protein sequence for TSC266:

Diqmtqspsamsasvgdrvtitcrasksiskylawfqqkpgkvpklrihsgstlqsgvpsrfsgsgsgteftltisslqpedfatyycqqh ieypwtfgqgtkveikrggggsggggsggggsqvqlvqsgaevkkpgasvkvsckasgytftdyymhwvrqapgqglewmgyf npyndytryaqkfqgrvtmtrdtsistaymelsslrsddtavyycarsdgyydamdywgqgttvtvsssepkssdkthtcppcpape aagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkay acavsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgqrhnnsslntgtqmaghspnsqvqlvesgggvvqpgrslrlsckasgytf trstmhwvrqapgqglewigyinpssaytnynqkfkdrftisadkskstaflqmdslrpedtgvyfcarpqvhydyngfpywgqgtp vtvssggggsggggsggggsaqdiqmtqspsslsasvgdrvtmtcsasssvsymnwyqqkpgkapkrwiydssklasgvparfsg sgsgtdytltisslqpedfatyycqqwsmpptfgggtklqitsss (SEQ ID NO:210)

- 114 042409

DNA sequence for ROR206:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacat cc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagc (SEQ ID NO:211)

Protein sequence for ROR206:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSmSNLAWFQQKP GKPPKLLIYRASNLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGVGAVSYRTSF GGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI NDYPITWVRQAPGKGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCPAPEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

- 115 042409

RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVESGGGV VQPGRSLRLSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRFTI SADKSKSTAFLQMDSLRPEDTGVYFCARPQVHYDYNGFPYWGQGTPVTVSSGGGGSG GGGSGGGGSAQDIQMTQSPSSLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWI YDSSKLASGVPARFSGSGSGTDYTLTISSLQPEDFATYYCQQWSRNPPTFGGGTKLQITS SS (SEQ ID NO:212)

DNA sequence for ROR207:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc aagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctccggctccggcaccgacttca ccctgaccatctcctccctgcagcccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttcg gcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatct gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgg a tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc

- 116 042409 agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagc (SEQ ID NO:213)

Protein sequence for ROR207:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKP GKPPKLLIYRASNLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGVGAVSYRTSF GGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI NDYPITWVRQAPGKGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCPAPEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVQSGPEV KKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVT ITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAP KRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKV EIKRSSS (SEQ ID NO:214)

DNA sequence for ROR208:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc cagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggctccggctccggcaccgacttc accctgaccatctcctccctggaggccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatc tgaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca acgactaccccatcacctgggtgcggcaggcccccggccagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca

- 117 042409 cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtcactga ggctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctggaatggattgga tacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttc ctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactg gggccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaagacatcc agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatga actggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtgg cagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacc cacccacgttcggaggggggaccaagctacaaattacatcctccagc (SEQ ID NO:215)

Protein sequence for ROR208:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKP GQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISSLEAEDVATYYCLGGVGAVSYRTS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI NDYPITWVRQAPGQGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCPAPEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVESGGGV VQPGRSLRLSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRFTI SADKSKSTAFLQMDSLRPEDTGVYFCARPQVHYDYNGFPYWGQGTPVTVSSGGGGSG GGGSGGGGSAQDIQMTQSPSSLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWI YDSSKLASGVPARFSGSGSGTDYTLTISSLQPEDFATYYCQQWSRNPPTFGGGTKLQITS SS (SEQ ID NO:216)

DNA sequence for ROR209:

atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgacatccagatgacccagtccccctcctccctg tccgcctccgtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggcctggttccagcagaagcccggc cagccccccaagctgctgatctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggctccggctccggcaccgacttc accctgaccatctcctccctggaggccgaggacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcggaggtggatccggcggtggcggctccggtggcggcggatc tgaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgtcctgcaccgcctccggctccgacatca

- 118 042409 acgactaccccatcacctgggtgcggcaggcccccggccagggcctggagtggatcggcttcatcaactccggcggctccacctggtac gcctcctgggtgaagggccggttcaccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccctgaagaccgaggac accgccgtgtactattgcgcccggggctactccacctactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctcga gtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaa ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag cctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttctggtggaggc ggttcaggcgga ggtggctccggcggtggcggatcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcctgggtcctcggtgaa ggtctcctgcaaggcttctggatataccttcagcagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagtggataggat acattaatcctagcagtgcttatactaattacaatcagaaattcaaggacagagtcacgattaccgcggacaaatccacgagcacagcctaca tggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaccccaagtccactatgattacaacgggtttccttactgg ggccaaggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatccggcggtggcggatcgggtggcggcgga tctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgcagtgccagctcaagtgtaagtt acatgaactggtatcagcagaaaccagggaaagcccctaagagatggatttatgactcatccaaactggcttctggggtcccatcaaggttc agcggcagtggatctgggacagattatactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtggagtcgt aacccacccactttcggcggagggaccaaggtggagatcaaacggtcctccagc (SEQ ID NO:217)

Protein sequence for ROR209:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKP GQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISSLEAEDVATYYCLGGVGAVSYRTS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI NDYPITWVRQAPGQGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCP APEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVQSGPEV KKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVT ITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSDIQMTQ SPSTLS AS VGDRVTMTC S AS S S VS YMNWYQQKPGKAP

- 119 042409

KRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKV EIKRSSS (SEQ ID NO:218)

DNA sequence for ROR231:

atggaggcacccgcccagctgctgttcttgttgctgctgtggctccctgataccaccggagacatccagatgacccaatccccttctagtctct ccgctagcgtcggagaccgcgtgaccatcaattgtcaagcatctcagagtattgacagcaatctcgcctggtttcagcagaagccaggaaa gccacccaagctcctgatttatagggctagcaacctggcttctggtgtccctagtaggttcagcggctctgggagtggcacagacttcaccct gaccattagtagtctgcagcccgaagatgtcgctacctattactgcctcggaggagtgggtgccgtttcttatcgcacctcatttggaggtggc accaaagtggagatcaaaggtggtggcggctccgggggtggcgggtcaggggggggagggtctggaggcggcggatcagaagttca gctggtggaatctggaggaggtctggtgcagccaggcaggtccctccggctgagctgcactgcatccggctctgacattaatgactaccct atcacctgggtgaggcaggcccccggtaaaggcctggagtggatcgggttcatcaattctggtggatctacttggtacgcaagctgggtga aaggacgcttcacaattagtagagacgactctaagtctatcgcatatctgcagatgaatagcttgaagacagaggacaccgccgtgtactatt gtgcaagaggatactccacttactaccgcgatttcaatatctggggccagggaaccctggtgacagtgtcctcgagtgagcccaaatcttctg acaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccc tcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacg gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac caggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaa agggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc aaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgg actccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcat gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggcggcggctccggcggcggcggcagcggc ggcggcggcagccccggatcccaggtgcagctggtgcagtctggtcctgaggtgaaaaagcctggctccagcgtgaaggtgtcctgcaa ggccagcggatacacctttagccggtccaccatgcattgggtgaggcaggctcctggacagggcctggagtggatcggctacatcaaccc cagcagcgcttataccaactacaatcagaagtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcctacatggagct gtccagcctgaggagcgaggataccgccgtgtactattgcgcccggccccaggtccattacgactacaacggcttcccctattggggcca gggaaccctggtgaccgtgtccagcggaggcggcggcagcggcggcggcggcagcggcggaggaggcagcggcggaggagg ctc cgacattcagatgacccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatgacctgcagcgccagcagctccgtgtc ctacatgaactggtaccagcagaagcccggcaaggctcccaagaggtggatttacgactccagcaagctggcctctggtgtccccagcag gttctctggtagcggcagcggcacagactacaccctgaccatctcctccctgcagcccgacgatttcgccacctactattgccagcagtggt cccggaatccccctacctttggcggcggcaccaaggtggagatcaagaggagctccagc (SEQ ID NO:219)

Protein sequence for ROR231:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKP GKPPKLLIYRASNLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGVGAVSYRTSF GGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI

- 120 042409

NDYPITWVRQAPGKGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCPAPEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVQSGPEV KKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVT ITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSDIQMTQ SPSTLS AS VGDRVTMTC S AS S S VS YMNWYQQKPGKAP KRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKV EIKRSSS (SEQ ID NO:220)

DNA sequence for ROR233:

atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgacaccacaggcgacatccagatgacccagtccccttcctccct gtccgctagcgtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactccaatctggcctggttccagcagaagcccgg acagccccccaagctgctgatttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatccggcagcggcaccgacttc accctgaccatctcctccctggaggccgaggatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcggacctccttcg gcggcggcaccaaggtggagatcaagggcggcggcggcagcggcggcggcggcagcggcggcggaggctccggcggcggcggc agcgaggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtccctgaggctgtcctgcacagccagcggctccgac atcaacgactaccccatcacctgggtgaggcaggctcctggccagggcctggaatggatcggctttatcaacagcggcggcagcacctg gtatgcttcctgggtgaagggccggttcaccattagcagggacgactccaagtccattgcctacctgcagatgaactccctgaagaccgag gacaccgccgtgtactactgcgccaggggctacagcacctattaccgggactttaacatctggggccagggcacactggtcaccgtgtcct cgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca agtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccagcccccatcga gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgt cttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggttccggaggcggcggctcc ggcggcggcggcagcggcggcggcggcagccccggatcccaggtgcagctggtgcagtctggtcctgaggtgaaaaagcctggctcc agcgtgaaggtgtcctgcaaggccagcggatacacctttagccggtccaccatgcattgggtgaggcaggctcctggacagggcctgga gtggatcggctacatcaaccccagcagcgcttataccaactacaatcagaagtttaaggaccgggtgaccatcaccgccgataagtccacc agcaccgcctacatggagctgtccagcctgaggagcgaggataccgccgtgtactattgcgcccggccccaggtccattacgactacaac ggcttcccctattggggccagggaaccctggtgaccgtgtccagcggaggcggcggcagcggcggcggcggcagcggcggaggagg

- 121 042409 cagcggcggaggaggctccgacattcagatgacccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatgacctgca gcgccagcagctccgtgtcctacatgaactggtaccagcagaagcccggcaaggctcccaagaggtggatttacgactccagcaagctg gcctctggtgtccccagcaggttctctggtagcggcagcggcacagactacaccctgaccatctcctccctgcagcccgacgatttcgcca cctactattgccagcagtggtcccggaatccccctacctttggcggcggcaccaaggtggagatcaagaggagctccagc (SEQ ID NO:221)

Protein sequence for ROR233:

MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKP GQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISSLEAEDVATYYCLGGVGAVSYRTS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDI NDYPITWVRQAPGQGLEWIGFINSGGSTWYASWVKGRFTISRDDSKSIAYLQMNSLKTE DTAVYYC ARGYSTYYRDFNIWGQGTLVT VS S SEPKS SDKTHTCPPCP APEAAGAPS VFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPGSQVQLVQSGPEV KKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVT ITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSDIQMTQ SPSTLS AS VGDRVTMTC S AS S S VS YMNWYQQKPGKAP KRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKV EIKRSSS (SEQ ID NO:222)

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Table 15 Sequence Summary SEQ ID NO

Purpose Description DNA SEQ ID NO Protein SEQ ID NO TSC311 or TSC312 Fc DRA222 1 2 TSC313 Fc H7L1 3 4 TSC314 Fc H7L4 5 6 TSC315 Fc H7L5 7 8 TSC316 Fc H8L1 9 10 TSC317 Fc H8L4 eleven 12 TSC318 Fc H8L5 13 14 TSC319 Fc H9L1 15 16 TSC320 Fc H9L4 17 18 TSC321 Fc H9L5 19 20 TSC322 Fc H10L1 21 22 TSC323 Fc H10L4 23 24 TSC324 Fc H10L5 25 26 TSC334 Fc H7L6 27 28 TSC335 Fc H7L7 29 thirty TSC336 Fc H7L8 31 32 TSC337 Fc H8L6 33 34 TSC338 Fc H8L7 35 36 TSC339 Fc H8L8 37 38 TSC340 Fc H10L6 39 40 TSC341 Fc H10L7 41 42 TSC342 Fc H10L8 43 44 TSC370 TSC342G27Y 45 46 TSC371 TSC342 M53I 47 48 TSC372 TSC342 I21M 49 50 TSC390 TSC370A9P 51 52 TSC391 TSC370 A9P M53I 53 54 TSC392 TSC370 A9P I21M 55 56 TSC393 TSC370 M53I I21M 57 58 TSC394 TSC370 A9P M53I I21M 59 60 TSC408 Anti-PSMA XTSC391 61 62

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TSC409 Anti-PSMA XTSC392 63 64 TSC410 Anti-PSMA XTSC393 65 66 TSC411 Anti-PSMA XTSC394 67 68 TSC471 Anti-PSMA XTSC456 205 206 TSC266 Anti-PSMA X DRA222 (huVL-VH 1071A4 scFv-Fc-DRA222 scFv) 209 210 CAS105 Ahth-CD37 X DRA222 69 70 Ahth-CD37 X TSC445 Ahth-CD37 X TSC394 71 72 Ahth-CD37 X TSC452 Ahth-CD37 X (TSC394 VL + DRA222 VH) 73 74 Ahth-CD37 X TSC453 Ahth-CD37 X (TSC394 VH + DRA222 VL) 75 76 Ahth-CD37 X TSC454 Ahth-CD37 X TSC394 E86D 77 78 Ahth-CD37 X TSC455 Ahth-CD37 X TSC394 F87Y 79 80 Ahth-CD37 X TSC456 Ahth-CD37 X TSC394 E86D F87Y 81 82 TSC455 TSC394 F87Y scFv (anti-POP scFv) 83 TSC456 TSC394 E86D F87Y scFv (anti-POP scFv) 84 DRA222 Ahth-CD3 scFv 85 TSC455 and TSC456 vh Variable heavy domain TSC455 and TSC456 86 DRA222 VH Variable heavy domain DRA222 87 TSC455 VL TSC455 variable light domain 88 TSC456 VL TSC456 variable light domain 89 DRA222 VL DRA222 variable light domain 90 Cris7 and DRA222 VH CDR1 (Kabat) Anti-POPs VH CDR1 91 Cris7 and DRA222 VH CDR2 (Kabat) Anti-POPs VH CDR2 92

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Cris7 and DRA222 VH CDR3 (Kabat) Ahtm-CD3 VH CDR3 93 Cris7 and DRA222 VL CDR1 (Kabat) Ahtm-CD3 VLCDR1 94 Cris7 and DRA222 VL CDR2 (Kabat) Ahtm-CD3 VLCDR2 95 Cris7 and DRA222 VL CDR3 (Kabat) Ahtm-CD3 VLCDR3 96 Cris7 and DRA222 VH CDR1 (IMGT) Ahtm-CD3 VH CDR1 199 Cris7 and DRA222 VH CDR2 (IMGT) Ahtm-CD3 VH CDR2 200 Cris7 and DRA222 VH CDR3 (IMGT) Ahtm-CD3 VH CDR3 201 Cris7 and DRA222 VL CDR1 (IMGT) Ahtm-CD3 VLCDR1 202 Cris7 and DRA222 VL CDR2 (IMGT) Ahtm-CD3 VLCDR2 203 Cris7 and DRA222 VL CDR3 (IMGT) Ahtm-CD3 VLCDR3 204 ROR133 Anti-RORl (Rll L7H15) X DRA222 97 98 ROR193 Anti-RORl (Rll L7H15) X TSC394 F87Y 99 100 ROR134 Anti-RORl (Rll L7H10) X DRA222 101 102 ROR189 Anti-RORl (Rll L7H10) X TSC394 E86D F87Y 103 104 ROR154 Anti-RORl (Rll L7H16) X DRA222 105 106 ROR185 Anti-RORl (Rll L7H15) X TSC394 E86D F87Y 107 108 ROR179 Anti-RORl (Rll L8H16) X DRA222 109 110 ROR186 Anti-RORl (Rll L8H16) X TSC394 E86D F87Y 111 112 ROR181 Anti-RORl (Rll L7H17) X DRA222 113 114 ROR191 Anti-RORl (Rll L7H17) X TSC394 E86D F87Y 115 116 ROR182 Anti-RORl (Rll L9H18) X DRA222 117 118

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Claims (37)

ROR192 Anti-RORl (Rll L9H18) X TSC394 E86D F87Y 119 120 ROR243 Humanized, codon-optimized anti-ROR1 X anti-POPs (parent anti-ROR1 molecule is ROR192) scFv-Fc-scFv 207 208 ROR206 ROR133 S40T G113R x DRA222 Fc ADCC-K322A-H111 in REE12.4 211 212 ROR207 ROR133 S40T G113R x TSC456 Fc ADCC-K322A-H111 in REE12.4 213 214 ROR208 ROR174 S40T G113R x DRA222 Fc ADCC-K322A-H111 in REE12.4 215 216 ROR209 ROR174 S40T G113R x TSC456 Fc ADCC-K322A-H111 in REE12.4 217 218 ROR231 Anti-RORl X anti-POPs 219 220 ROR233 Anti-RORl X anti-POPs 221 222 Chimeric bispecific anti-CD123 (VLVH) x anti-POPs (TSC456) scFv-Fc-scFv 197 Chimeric bispecific anti-CD123 (VHVL) x anti-POPs (TSC456) scFv-Fc-scFv 198 CLAIM 1. A CD3 binding polypeptide comprising a CD3 binding domain that specifically binds to human CD3 and which contains a humanized immunoglobulin light chain variable region and a humanized immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region comprises the LCDR1 amino acid sequence shown in SEQ ID NO: 94, the LCDR2 amino acid sequence shown in SEQ ID NO: 95, and the LCDR3 amino acid sequence shown in SEQ ID NO: 96, wherein the immunoglobulin heavy chain variable region contains the HCDR1 amino acid sequence shown in SEQ ID NO: 91 , the amino acid sequence of HCDR2 shown in SEQ ID NO: 92, and the amino acid sequence of HCDR3 shown in SEQ ID NO: 93; wherein the amino acid residue at position 9, according to the IMGT numbering system of the immunoglobulin heavy chain variable region, is proline; and wherein the amino acid residue at position 21 according to the IMGT numbering system of the immunoglobulin light chain variable region is methionine; and wherein the amino acid residue at position 87 according to the IMGT numbering system of the immunoglobulin light chain variable region is tyrosine. 2. CD3-binding polypeptide according to claim 1, characterized in that: (a) the immunoglobulin light chain variable region contains an amino acid sequence at least 96% identical to SEQ ID NO: 88, and the immunoglobulin heavy chain variable region contains an amino acid sequence at least 96% identical to SEQ ID NO: 86; or (b) the immunoglobulin light chain variable region contains an amino acid sequence at least 96% identical to SEQ ID NO: 89, and the immunoglobulin heavy chain variable region contains an amino acid sequence at least 96% identical to SEQ ID NO: 86. 3. CD3-binding polypeptide according to claim 1 or 2, characterized in that: (a) the immunoglobulin light chain variable region comprises SEQ ID NO: 88 and the immunoglobulin heavy chain variable region comprises SEQ ID NO: 86; or - 126 042409 (b) the immunoglobulin light chain variable region contains SEQ ID NO: 89 and the immunoglobulin heavy chain variable region contains SEQ ID NO: 86. 4. A CD3-binding polypeptide according to claim 1, characterized in that the amino acid residue at position 52 according to the IMGT numbering system of the immunoglobulin light chain variable region is arginine and/or the amino acid residue at position 53 according to the IMGT variable region numbering system The light chain immunoglobulin is tryptophan. 5. The CD3 binding polypeptide of claim 1, wherein the amino acid residue at position 27, according to the IMGT numbering system of the immunoglobulin heavy chain variable region, is tyrosine. 6. The CD3 binding polypeptide of claim 1, wherein the amino acid residue at position 53, according to the IMGT numbering system of the immunoglobulin heavy chain variable region, is isoleucine. 7. The CD3 binding polypeptide of claim 1, wherein the amino acid residue at position 86, according to the IMGT numbering system of the immunoglobulin light chain variable region, is aspartic acid. 8. A CD3 binding polypeptide according to any one of the preceding claims, characterized in that the CD3 binding domain comprises SEQ ID NO: 83 or SEQ ID NO: 84. 9. A CD3 binding polypeptide according to any one of the preceding claims, wherein the CD3 binding domain is a single chain variable fragment (scFv). 10. The CD3 binding polypeptide of claim 9, wherein said scFv contains a linker between the heavy chain variable region and the light chain variable region of said scFv, and said linker comprises the amino acid sequence QRHNNSSLNTGTQMAGHSPNS (SEQ ID NO: 148). 11. The CD3 binding polypeptide of claim 9 or 10, wherein the heavy chain variable region of said scFv is amino-terminal to the light chain variable region of said scFv. 12. CD3-binding polypeptide containing: (i) a first binding domain, which is a CD3 binding domain that specifically binds to human CD3 and comprises a humanized immunoglobulin light chain variable region and a humanized immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region comprises the amino acid sequence LCDR1 represented by in SEQ ID NO: 94, the LCDR2 amino acid sequence shown in SEQ ID NO: 95, and the LCDR3 amino acid sequence shown in SEQ ID NO: 96, and the immunoglobulin heavy chain variable region contains the HCDR1 amino acid sequence shown in SEQ ID NO: : 91, the amino acid sequence of HCDR2 shown in SEQ ID NO: 92 and the amino acid sequence of HCDR3 shown in SEQ ID NO: 93; wherein the amino acid residue at position 9, according to the IMGT numbering system of the immunoglobulin heavy chain variable region, is proline; and wherein the amino acid residue at position 21 according to the IMGT numbering system of the immunoglobulin light chain variable region is methionine; and wherein the amino acid residue at position 87 according to the IMGT numbering system of the immunoglobulin light chain variable region is tyrosine; and (ii) a second binding domain. 13. The CD3-binding polypeptide of claim 12, wherein said CD3-binding polypeptide contains, in amino-terminal to carboxy-terminal order: (i) a second binding domain, (ii) a hinge region, (iii) a constant region immunoglobulin, (iv) a carboxy-terminal linker, and (v) a CD3 binding domain. 14. CD3 binding polypeptide according to claim 13, characterized in that said immunoglobulin constant region contains a human IgG1 CH2 domain containing substitutions L234A, L235A, G237A and K322A according to the EU numbering system. 15. CD3-binding polypeptide according to claim 12 or 14, characterized in that: (i) the CD3 binding domain comprises: (a) an immunoglobulin light chain variable region comprising LCDR1, LCDR2 and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; and (ii) the second binding domain comprises: (a) an immunoglobulin light chain variable region comprising LCDR1, LCDR2 and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2 and HCDR3. 16. A CD3 binding polypeptide according to any one of claims 13-15, characterized in that the hinge region is derived from the hinge region of an immunoglobulin. 17. CD3 binding polypeptide according to any one of claims 13-16, characterized in that the carboxy-terminal linker contains or consists of SEQ ID NO: 196. - 127 042409 18. A CD3-binding polypeptide according to any one of claims 13-17, characterized in that the immunoglobulin constant region contains immunoglobulin CH2 and CH3 domains from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD. 19. A CD3 binding polypeptide according to any one of claims 12-18, wherein the CD3 binding polypeptide induces redirected T cell cytotoxicity (PTCC). 20. The CD3 binding polypeptide of claim 19, wherein the CD3 binding polypeptide induces PTCC with an EC ₅₀ of about 30 pM or less. 21. A CD3 binding polypeptide according to any one of claims 12-20, wherein the second binding domain is a single chain variable fragment (scFv). 22. A CD3 binding polypeptide according to any one of claims 12-21, characterized in that the second binding domain binds or interacts with a tumor associated antigen. 23. The CD3 binding polypeptide of claim 22, wherein said CD3 binding polypeptide induces T-cell dependent lysis of cells expressing the tumor associated antigen. 24. CD3-binding polypeptide according to claim 22 or 23, characterized in that the tumor-associated antigen is selected from the group consisting of PSMA, CD19, CD20, CD37, CD38, CD123, Her2, ROR1, RON, A33 glycoprotein antigen (gpA33) and CEA. 25. An isolated nucleic acid molecule encoding a CD3 binding polypeptide according to any one of claims 1 to 24, or a portion of said CD3 binding polypeptide that comprises a humanized immunoglobulin light chain variable region and a humanized immunoglobulin heavy chain variable region. 26. An expression vector containing a nucleic acid segment encoding a CD3 binding polypeptide according to any one of claims 1 to 24, wherein the nucleic acid segment is operably linked to regulatory sequences suitable for expression of the nucleic acid segment in the host cell. 27. Recombinant host cell containing the expression vector of claim 26. 28. A method for producing a CD3-binding polypeptide, comprising culturing a recombinant host cell containing the expression vector of claim 26 under conditions that express the nucleic acid segment and thereby obtaining a CD3-binding polypeptide. 29. The method of claim 28, further comprising isolating the CD3 binding polypeptide. 30. A pharmaceutical composition for inhibiting tumor growth in a subject in need thereof, comprising a CD3 binding polypeptide according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier, diluent or excipient. 31. A method for inducing redirected T-cell cytotoxicity (PTCC) against a cell expressing a tumor-associated antigen, comprising contacting said cell expressing a tumor-associated antigen with a CD3-binding polypeptide according to any one of claims 1-24, said contacting occurring under conditions in which PTCC is induced against a cell expressing a tumor-associated antigen. 32. A method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD3 binding polypeptide according to any one of claims 1-24 or a composition according to claim 30. 33. A method of treating cancer or an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD3 binding polypeptide according to any one of claims 1-24 or a composition according to claim 30. 34. The method of claim 33, wherein the cancer is prostate cancer, colorectal cancer, renal cell carcinoma, bladder cancer, salivary gland cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, breast cancer, melanoma, cancer adrenal glands, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute myelocytic leukemia (AML), B-lymphoid leukemia, plasmacytoid dendritic cell blast neoplasm (PPDC), or hairy cell leukemia. 35. The method of claim 34, wherein said breast cancer is triple negative breast cancer. 36. A CD3 binding protein which is a dimer of two identical polypeptides, each polypeptide being a CD3 binding polypeptide according to any one of claims 1-24. 37. A CD3 binding polypeptide according to any one of claims 1 to 24, wherein the CD3 binding polypeptide exhibits no or minimal antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity. - 128 -