T cell binding compositions and methods

Abstract

The present disclosure relates generally to binding proteins comprising an antigen binding site, a T cell receptor binding site, and a T cell costimulatory molecule binding site. The present disclosure also relates to pharmaceutical compositions comprising such binding proteins, nucleic acid molecules encoding such binding proteins, and vectors comprising such nucleic acid molecules. The present disclosure also relates to methods of using such binding proteins and pharmaceutical compositions to treat a disorder or condition, binding proteins and pharmaceutical compositions for use in treating a disorder or condition, and uses of such binding proteins and pharmaceutical compositions for the manufacture of a medicament for treating a disorder or condition.

Description

[0001] This application is a divisional application of the invention patent application filed on October 9, 2024, with application number 202480064344.0 and entitled "T-cell binding composition and method".

[0002] This application claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 589,297, filed October 10, 2023, and U.S. Provisional Patent Application Serial No. 63 / 658,583, filed June 11, 2024. The entire contents of each of these applications are hereby incorporated herein by reference.

[0003] Sequence list declaration The sequence list in computer-readable form is submitted electronically with this application and is incorporated herein by reference in its entirety. The sequence list is contained in a file named “23-1331-WO.xml” created on September 22, 2024, and is 276,466 bytes in size. Technical Field

[0004] This disclosure generally relates to binding proteins comprising antigen-binding sites, T-cell receptor binding sites, and T-cell co-stimulatory molecule binding sites. This disclosure also relates to pharmaceutical compositions comprising such binding proteins, nucleic acid molecules encoding such binding proteins, and carriers comprising such nucleic acid molecules. This disclosure further relates to methods of treating a condition or symptom using such binding proteins and pharmaceutical compositions, binding proteins and pharmaceutical compositions for use in treating a condition or symptom, and the use of such binding proteins and pharmaceutical compositions in the manufacture of medicaments for treating a condition or symptom. Background Technology

[0005] Recruiting T cells to engage in cytotoxic activity to destroy tumor cells is a promising but complex cancer treatment strategy. The development of CD3-based bispecific T-cell connectives (TCEs) as cancer therapeutics has been ongoing for the past 30 years. TCEs simultaneously bind to tumor-associated antigens (TAAs) and differentiation cluster 3 (CD3) on T cells to form T-cell receptor (TCR)-independent artificial immune synapses, thereby bypassing human leukocyte antigen (HLA) restrictions and inducing T-cell activation and tumor cell lysis.

[0006] The first generation of TCEs was a simple bispecific T-cell connective (BiTE) consisting of two tandem single-stranded variable fragments (scFvs), including a strong CD3-binding arm and a TAA-binding domain. To date, only one BiTE has been approved by the Food and Drug Administration, blinatumomab, which targets CD3 (using the Orthoclone OKT3 antibody) and differentiation cluster 19 (CD19). The strong in vitro cytolytic activity observed during the development of BiTEs has generated interest surrounding their potential use in cancer treatment. However, the unexpected severe cytokine release syndrome (CRS) observed in clinical settings has somewhat diminished this interest. (Teachey et al., “Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy”, Blood 121: 5154-57 (2013). Another observed drawback of BiTE forms is their very short half-life and poor manufacturability.) Ellerman, “Bispecific T cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety”, Methods 154: 102-17 (2019).

[0007] Second-generation TCEs contain crystallizable fragment (Fc) domains that can be modified to confer extended half-life and mutated to eliminate Fc receptor (FcR) binding, exhibiting improved manufacturability. (Vafa et al., “Perspective: Designing T cell Engagers With Better Therapeutic Windows”, Front. Oncol. 10: 446 (2020).) However, these molecules still contain high-affinity CD3-binding domains associated with the induction of neurotoxicity and CRS in clinical settings. Recent efforts have focused on developing CD3-binding domains with reduced affinity, hoping to maintain potent T cell activation while significantly reducing the release of associated cytokines. (Trinklein et al., “Efficient tumor killing and minimal cytokine release with novel T cell agonist bispecific antibodies”, MAbs 11:639-52 (2019).

[0008] Importantly, both CD3-based BiTE and the novel immunoglobulin G (IgG) form of TCE bind to and activate both differentiation cluster 4 (CD4) and differentiation cluster 8 (CD8) T cells, potentially engaging with unfavorable T cells, such as regulatory T cells (Tregs), which have been shown to potentially reduce the cytolytic activity of CD8 T cells. (Duell et al., “Frequency of regulatory T cells determines the outcome of the T cell-engaging antibody blinatumomab in patients with B-precursor ALL”, Leukemia 31: 2181-90 (2017).)

[0009] While T-cell adaptor molecules offer promise, therapeutic applications have so far faced challenges. There is a need in the field for improved T-cell binding proteins with increased activity and reduced off-target effects. Summary of the Invention

[0010] In a first aspect, this disclosure provides a binding protein comprising four polypeptide chains forming two tumor-associated antigen (TAA) binding sites, a T-cell receptor binding site, and a T-cell co-stimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the following formula: V L -C L Furthermore, the third polypeptide chain has a structure represented by the following formula: VH1 -C H1- V HHa -Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -V HHb -Fc b The first and third polypeptides form the first TAA binding site of the two TAA binding sites, and the second and fourth polypeptides form the second TAA binding site of the two TAA binding sites, wherein: V L It is the variable domain of the immunoglobulin light chain, and V H1 These are the variable domains of the immunoglobulin heavy chain, which together form the TAA-binding domain that specifically binds to tumor-associated antigens; C L It is the constant domain of the immunoglobulin light chain; C H1 It is the CH1 heavy chain constant domain of immunoglobulin; V HHa It is a single-stranded variable domain that specifically binds to the T-cell receptor; V HHb It is a single-stranded variable domain that specifically binds to T cell co-stimulatory molecules; Fc a It is C H2a and C H3a Immunoglobulin heavy chain constant domain; and Fc b It is C H2b and C H3b Immunoglobulin heavy chain constant domain.

[0011] In a second aspect, this disclosure provides novel binding proteins. Attached Figure Description

[0012] The accompanying drawings are included to provide a further understanding of the methods and compositions of this disclosure, and are incorporated in and constitute a part of this disclosure. The drawings illustrate one or more aspects of this disclosure and, together with the description, serve to explain the principles and operation of this disclosure.

[0013] Figure 1 A to Figure 1 D: Figure 1 A shows the in vitro cell lysis activity of LM1486 and CD3 adaptors that bind to CD20 monovalently or bivalently. Figure 1 B shows the corresponding EC for in vitro cell lysis assay. 50 . Figure 1 C shows the corresponding CD4T cell activation profile, with the percentage of cells expressing CD25 indicated. Figure 1 D shows the corresponding CD8T cell activation profile, with the percentage of cells expressing CD25 indicated.

[0014] Figure 2 A to Figure 2 D shows that for LM1486, IL-6 ( Figure 2 A) IL-10 Figure 2 B), TNF-α Figure 2 C) and IL-17A Figure 2 The cytokine release profile of D).

[0015] Figure 3 The in vitro cell lysis activity of LM1486, a different CD20+ B cell line, incubated with PBMCs at an E:T ratio of 5:1 is shown.

[0016] Figure 4 A to Figure 4 E illustrates the structure of the LM1486 ( Figure 4 A) and the structure of a modified binding protein having a null binding domain that replaces the anti-CD8 domain. Figure 4 B) or the structure of the modified binding protein that replaces the ineffective binding domain of the anti-TCR binding domain. Figure 4 C). Figure 4 D shows the in vitro cell lysis activity of LM1486 and control binding proteins TENG0501 and TENG0502. Figure 4 E shows the corresponding T cell activation profile, with the percentage of CD4 and CD8 T cells expressing CD25 shown.

[0017] Figure 5 A to Figure 5 E illustrates the structure of the LM1486 ( Figure 5 A) and the structure of a modified binding protein having a null binding domain that replaces the anti-CD20 domain on the anti-CD8 arm. Figure 5 B) or a modified binding protein structure having a null binding domain that replaces the anti-CD20 binding domain on the anti-TCR arm. Figure 5 C). Figure 5 D shows the in vitro cell lysis activity of LM1486 and control binding proteins TENG0499 and TENG0500. Figure 5 E shows the corresponding T cell activation profile, with the percentage of CD4 and CD8 T cells expressing CD25 shown.

[0018] Figure 6 A to Figure 6 D represents the tumor burden, total flux (p / s), presented as a PBS-treated group ( Figure 6 A) 0.8 mg / kg of LM1486 ( Figure 6 B), 0.08 mg / kg of LM1486 ( Figure 6 C) and 0.008 mg / kg of LM1486 ( Figure 6 D).

[0019] Figure 7 A to Figure 7 C shows tumor growth inhibition compared to the conventional CD3 adaptor in a fully humanized in vivo model with subcutaneous B-cell tumors. Figure 7 A) Survival percentage ( Figure 7 B) and percentage of weight loss ( Figure 7 C).

[0020] Figure 8 A to Figure 8 F shows the antitumor efficacy of LM1486 in an in vivo model of diffuse DLBCL: total flux ( Figure 8 A), TNF-α Figure 8 B), IL-10 Figure 8 C) IL-2 Figure 8 D), IL-17A Figure 8 E) and % weight loss ( Figure 8 F).

[0021] Figure 9 A to Figure 9 F shows the in vitro cell lysis activity of the LM1486 and LM1486-2 connectors. Figure 9 A and Figure 9 D shows the cytotoxicity profiles of two different PBMC donors within the T-cell adaptor concentration range. Figure 9 B. Figure 9 C and Figure 9 E, Figure 9 F shows the relevant CD4 and CD8 T cell activation profiles for the two donors, expressed as a percentage of CD25 surface expression.

[0022] Figure 10 A to Figure 10 C shows the binding of LRC150016 to various cell lines; combined LRC15pos cell lines ( Figure 10 A) Saos-2 Figure 10 B) and RPMI Figure 10 C).

[0023] Figure 11 A to Figure 11 B shows that the LRC150016 adaptor mediates T cell effector function in an antigen-dependent (LRRC15) manner. Figure 11 A shows the in vitro cell lysis activity of LRC150016 against the target cell lines LRRC15pos (Saos-2 WT) and LRRC15neg (Saos-2 KO and A431-WT). Figure 11B shows the cytokine profile measured in the supernatant of an in vitro T cell lysis assay using a multiplex cytokine assay (e.g., ...). Figure 11 (As outlined in section A).

[0024] Figure 12 A to Figure 12 C shows the in vitro cell lysis and CD8-biased T cell activation profile induced by LRC150016 compared to the comparative CD3 TCE. Figure 12 A shows the in vivo and in vitro cell lysis activity mediated by LRC150016 or the comparative CD3 TCE. Figure 12 B shows the percentage of CD8 mediated by LRC150016 or the comparative CD3 TCE, as measured by expression on the CD25 surface. + T cell activation. Figure 12 C shows the CD4+ T cell activation mediated by LRC150016 or the comparative CD3 TCE, as measured by the percentage expressed on the CD25 surface.

[0025] Figure 13 A to Figure 13 D shows the in vitro cytokine profile of LRC150016 compared to the comparative drug CD3 TCE: TNFα ( Figure 13 A) IL-6 ( Figure 13 B), IL-10 Figure 13 C) and IL-2 Figure 13 D).

[0026] Figure 14 A to Figure 14 B shows a series of cell lines expressing different levels of LRRC15, and the specific in vitro cell lysis activity of LRC150016. Figure 14 A shows E max ,and Figure 14 B shows EC 50 .

[0027] Figure 15 A diagram showing the LRC150016 binding protein.

[0028] Figure 16 A to Figure 16 E shows the in vitro cell lysis induced by LM1653 against hepatocellular carcinoma (HCC) cell lines expressing different levels of GPC3, compared to the conventional GPC3xCD3 adapter. Figure 16 A shows GPC3 expression across four HCC cell lines. Figure 16 B to Figure 16 E shows LM1653 and monovalent GPC3xCD3 binders targeting the HCC cell line HepG2 ( Figure 16 B), Hep3B Figure 16 C), Huh-7 Figure 16 D) and PLC / PRF / 5 ( Figure 16 E) in vitro cell lysis activity.

[0029] Figure 17 A to Figure 17 D shows the results in the HCC cell line HepG2 ( Figure 17 A), Hep3B Figure 17 B), Huh-7 Figure 17 C) and PLC / PRF / 5 ( Figure 17 Activation profiles of CD8+ T cells (top) and CD4+ T cells (bottom) induced by LM1653 and the monovalent GPC3xCD3 adaptor in cytotoxicity assay D).

[0030] Figure 18 A to Figure 18 D shows the in vitro cytokine release profile of LM1653 cells using the HCC Hep3B cell line as target cells at an E:T ratio of 10:1 in an in vitro cell lysis assay, compared to the conventional GPC3xCD3 connector: TNF-α ( Figure 18 A) IL-10 Figure 18 B), IL-2 Figure 18 C) and IFN-γ Figure 18 D).

[0031] Figure 19 A to Figure 19 H shows the non-specific activity of LM1653 and the conventional GPC3xCD3 adaptor in the plate binding assay. CD8 and CD4 T cell activation profiles (defined as the percentage of parental cells expressing CD25) are plotted on [data missing]. Figure 19 A and Figure 19 B. Histograms represent mean ± SEM; *, p < 0.05, two-way ANOVA. IL-6 release was measured in the supernatant at 48 hours using multiple determination (Luminex). Figure 19 C), TNF-α Figure 19 D), IFN-γ Figure 19 E), IL-2 Figure 19 F), IL-10 Figure 19 G) and IL-17 Figure 19 H). Results from a representative donor PBMC are shown.

[0032] Figure 20 A to Figure 20J shows the in vitro cell lysis activity of purified CD4 T cells (top) and purified CD8 T cells (bottom) used in the Xcelligence assay, LM1653, and the monovalent GPC3xCD3 adaptor. Figure 20 A to Figure 20 B) and cytokine secretion ( Figure 20 C to Figure 20 J).

[0033] Figure 21 A to Figure 21 B shows the progression from HCC stage II ( Figure 21 A) and Stage IIIB ( Figure 21 B) Patient-derived tumor-infiltrating lymphocytes against GPC3 + LM1653-dependent killing of Hep3B cell line.

[0034] Figure 22 A to Figure 22 D shows the LM1653 and monovalent GPC3xCD3 connector for the non-small cell lung cancer line NCI-H661 ( Figure 22 A) and NCI-H2172 ( Figure 22 B) and ovarian cancer cell line OV-90 ( Figure 22 C) and Kuramochi ( Figure 22 D) in vitro cell lysis activity.

[0035] Figure 23 A to Figure 23 F shows comparable in vitro cell lysis and T cell activation profiles induced by LM1653 and LM1653-2 (same sequence as LM1653, but different batches). Figure 23 A to Figure 23 On day 4, cell lysis activity was measured using PBMCs at a ratio of 10:1 in the XCELLIGENCE system (n = 1 PBMC donor). The table shows the cell lines Hep3B (GPC3+). Figure 23 A) and Huh-7 Figure 23 B) Corresponding EC 50 And lethality E max Using Hep3B ( Figure 23 C to Figure 23 D) and Huh-7 cell line ( Figure 23 E to Figure 23 F) T cell activation profiles were determined in a cytotoxicity assay. The percentages of CD8 (solid symbol) and CD4 (hollow symbol) T cells expressing CD25 were reported.

[0036] Figure 24 A to Figure 24 B shows that LM1653 is designed for GPC3.高 and GPC3 低 In vivo antitumor efficacy of HCC xenograft model. Figure 24 A shows 5e06 Hep3B cells expressing high levels of GPC3. On day 21, the animals were humanized with 10e06 panT cells expanded in vitro. Tumor burden was measured over time and reported as mean ± SEM. ‡ 40% were tumor-free; **, p<0.01 and ***, p<0.001, two-way ANOVA. Figure 24 B shows 5e06 PLC / PRF / 5 cells expressing low levels of GPC3. Tumor burden was measured over time and reported as mean ± SEM. **, p < 0.0001, two-way ANOVA. Dashed lines indicate TCE administration.

[0037] Figure 25 A to Figure 25 C shows LM1653 (GPC3 TITAN) with EC 50 :8 nM binds to human liver cancer cells Hep3B ( Figure 25 A) and EC 50 :2 nM binds to human liver cancer cells HepG2 ( Figure 25 B). GPC3 TITAN with EC 50 6 nM binds to human PBMCs ( Figure 25 C).

[0038] Figure 26 A and Figure 26 B describes the comparative CD3xCD20 T cell connector.

[0039] Figure 27 A to Figure 27 F describes LM1486 (CD20 TITAN) in patients with SLE ( Figure 27 A, Figure 27 C and Figure 27 E) and myositis ( Figure 27 B. Figure 27 D and Figure 27 Excellent B-cell depletion in whole blood PBMCs of patients with F) Figure 27 A and Figure 27 B) CD4 T cell retention activity ( Figure 27 C and Figure 27 D) and selective CD8+ T cell activation ( Figure 27 E and Figure 27 F).

[0040] Figure 28A and 28B compared the cytokine release of different concentrations of motuzumab and LM1486 (CD20 TITAN) in a nonspecific T cell activation assay: IL-17A ( Figure 28 A) and TNFα Figure 28 B).

[0041] Figure 29 A humanized mouse model of B cell depletion was described.

[0042] Figure 30 A to Figure 30 F depicts the spleen in humanized mouse models treated with solvent, NIP228 (control), or different single doses of LM1486 (CD20TITAN). Figure 30 A) Blood ( Figure 30 B) Bone marrow Figure 30 C) Kidney ( Figure 30 D) Thymus ( Figure 30 E) and lymph nodes ( Figure 30 B cell depletion in F).

[0043] Figure 31 A to Figure 31 B shows the potent in vitro cell lysis of LM1953. Figure 31 A shows the in vitro cell lysis activity of LM1953 against C4-2 cells and the corresponding EC50. 50 . Figure 31 B shows the in vitro cell lysis activity of LM1953 on 22Rv1 cells and the corresponding EC50. 50 LM1953 induced a CD8-biased T cell activation profile.

[0044] Figure 32 A to Figure 32 B shows the assay of C4-2 cell lysis ( Figure 32 A) and 22Rv1 cell lysis assay Figure 32 The corresponding CD4 and CD8 T cell activation profiles in B) are shown as the percentage of cells expressing CD25 on their cell surface.

[0045] Figure 33 A to Figure 33 D shows the effect of LM1953 on IL-6 in C4-2 cells ( Figure 33 A) and TNF-α Figure 33 C), and on 22Rv1 cells, IL-6 ( Figure 33 B) and TNF-α Figure 33 D) In ​​vitro cytokine release profile.

[0046] Figure 34This demonstrates the in vivo antitumor efficacy of LM1953 against the STEAP2+ prostate cancer xenograft model 22Rv1.

[0047] Figure 35 A to Figure 35 C shows that the LRRC15 variant preferentially activates CD8+ T cells and has functional activity. Figure 35 A) is cytotoxic. Figure 35 B), and IL-6 concentration ( Figure 35 C).

[0048] Figure 36 The binding of the LRRC15 TCE variant to EphB6-expressing As293 cells is shown.

[0049] Figure 37 A to Figure 37 C shows the bone marrow ( Figure 37 A) and spleen ( Figure 37 B) Three days after a single TCP-42197 (BCMA TCE) treatment, plasmablasts were depleted in mice. Serum human IgG levels were significantly lower in the TCP-42197-treated group compared to the isotype control group. Figure 37 C).

[0050] Figure 38 The results show that TPP-42197 (BCMA TCE) plasmablasts are highly depleted compared to the comparison T.

[0051] Figure 39 A to Figure 39 B shows TPP-42197 (BCMA TCE) relative to CD4+ T cells ( Figure 39 B) Selective activation of CD8+ T cells ( Figure 39 A).

[0052] Figure 40 A to Figure 40 F shows that the cytokine profile of TPP-42197 (BCMA TCE) is superior to that of the comparative substance T, with overall lower cytokine secretion, especially those cytokines involved in CRS events: IL-17A ( Figure 40 A) IL-22 Figure 40 B), IL-10 Figure 40 C), IL-6 Figure 40 D), TNFα Figure 40 E) and IFNγ Figure 40 F).

[0053] Figure 41 A to Figure 41E showed that, compared with the conventional GPC3xCD3 connector, LM1653-2 induced significant tumor growth inhibition in mice carrying Hep3B tumors, without significant secretion of related cytokines. Figure 41 A shows the tumor burden, which was monitored twice weekly by bioluminescence imaging (BLI) until day 24, i.e., 2 days after treatment. Serum cytokine concentrations for each treatment group are shown: IFN-γ ( Figure 41 B), TNFα Figure 41 C), IL-10 Figure 41 D) and IL-2 ( Figure 41 E).

[0054] Figure 42 A to Figure 42 E showed that, compared with solvent-treated animals, LM1653-3 (same sequence as LM1653 but different batch) induced significant tumor growth inhibition in mice carrying NCI-H661 lung tumors without significant secretion of related cytokines. Figure 42 A shows the tumor burden, which was monitored twice weekly by bioluminescence imaging (BLI). Serum cytokine concentrations for each treatment group are shown: IFNγ ( Figure 42 B), TNFα Figure 42 C), IL-10 Figure 42 D) and IL-2 ( Figure 42 E).

[0055] Figure 43 A to Figure 43 E shows that administration of LM1653 to cynomolgus monkeys resulted in a limited increase in transient CD8+ T cell attachment and circulating cytokine levels. Figure 43 A shows the absolute count of cytotoxic CD8+ T cells in the blood during treatment, reported as mean ± SD for each group. Dosage: 1 mg / kg / week IV (Δ), 6 mg / kg / week IV ( ), or 6 mg / kg / week SC (◇), followed by a 2-week recovery period. Animals that received only the solvent in either IV or SC form (○) served as controls. Figure 43 B to Figure 43 E shows serum IL-10 levels over time during treatment. Figure 43 B), IL-6 Figure 43 C), MCP-1 Figure 43 D) and MIP-1b Figure 43 E) The level was determined using multiple determinations (MSD) and reported as the mean for each treatment group. Solid and dashed lines represent values ​​obtained for males and females, respectively.

[0056] Figure 44A schematic diagram of a cynomolgus monkey dosage study of LM1486 (CD20 TITAN) is shown.

[0057] Figure 45 A to Figure 45 B indicates the result of B cell depletion in blood and tissues. Figure 45 A shows the results of circulating CD19+ B cells, and Figure 45 B shows tissue-specific CD19+ B cells in the spleen, bone marrow, and lymph nodes.

[0058] Figure 46 A to Figure 46 C shows the CD8+ after the first dose. Figure 46 A) and CD4+ Figure 46 B) The absolute circulating level of T cells and the absolute circulating level of activated CD8 / CD4 (CD25+) Figure 46 C). LM1486 and CD20 TITAN are interchangeable.

[0059] Figure 47 A to Figure 47 C shows the timeline from day -14 to day 16, IL-6 ( Figure 47 A), TNFα Figure 47 B) and IFNγ Figure 47 Group C) mean plasma cytokine profile.

[0060] Diagrams and amino acid sequences of various T-cell adaptors illustrated in this article are shown. LM1486-2(CD20)( Figure 48A ), LM1653(GPC3)( Figure 48B ), TPP-42197(BCMA)( Figure 48C ), LM1953(STEAP2)( Figure 48D ), TPP-46956(LRRC15)( Figure 48E ), TPP-46957(LRRC15)( Figure 48F ), TPP-46958(LRRC15)( Figure 48G ), TPP-46959(LRRC15)( Figure 48H ), TPP-46960(LRRC15)( Figure 48I ), TPP-47826(LRRC15)( Figure 48J ), TPP-49058(LRRC15)( Figure 48K ) and LM1486 ( Figure 48L ). Detailed Implementation

[0061] This disclosure generally relates to binding proteins comprising antigen-binding sites, T-cell receptor binding sites, and T-cell co-stimulatory molecule binding sites. This disclosure also relates to pharmaceutical compositions comprising such binding proteins, nucleic acid molecules encoding such binding proteins, and carriers comprising such nucleic acid molecules. This disclosure further relates to methods of treating a condition or symptom using such binding proteins and pharmaceutical compositions, binding proteins and pharmaceutical compositions for use in treating a condition or symptom, and the use of such binding proteins and pharmaceutical compositions in the manufacture of medicaments for treating a condition or symptom.

[0062] It should be understood that the specific aspects of this disclosure described herein are not limited to those presented and may vary. It will also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting unless specifically defined herein. Furthermore, as those skilled in the art will recognize, the specific aspects disclosed herein can be combined with other aspects disclosed herein in a non-limiting manner.

[0063] Unless otherwise stated or apparent from the context and from the understanding of one of ordinary skill in the art, values ​​expressed herein as ranges may be assumed to be any particular value or subrange within the ranges described in different aspects of this disclosure, up to one-tenth of the unit of the lower limit of the range, unless the context expressly specifies otherwise.

[0064] Throughout this disclosure, unless the context specifically indicates otherwise, the terms “comprise” and “include” and their variations (e.g., “comprises”, “comprising”, “includes”, and “including”) will be understood to indicate that a group of stated components, features, elements, or steps is included, but does not exclude any other components, features, elements, or steps or groups of components, features, elements, or steps.

[0065] As used herein, unless the context clearly indicates otherwise, the singular forms “a” and “the” include multiple referents. Unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms.

[0066] The percentages disclosed herein may differ quantitatively from the disclosed values ​​by ±10%, 20%, or 30%, and will still remain within the range of intended disclosure.

[0067] As used herein, ranges and quantities can be expressed as “about” a specific value or range. The term “about” also includes precise quantities. For example, “about 5%” means “about 5%” as well as “5%”. The term “about” can also refer to ±10% of a given value or range of values. Therefore, about 5% also means, for example, 4.5%–5.5%.

[0068] As used herein, the terms “or” and “and / or” can be used to describe multiple components that are combined or mutually exclusive. For example, “x, y and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y and z”, “(x and y) or z”, “x or (y and z)” or “x or y or z”.

[0069] As used in this disclosure, unless otherwise stated, all technical and scientific terms should be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0070] As used herein, the term “binding protein” refers to a non-naturally occurring (or recombinant) molecule containing multiple polypeptide chains that form at least one antigen-binding site.

[0071] "Recombinant" molecules are molecules that are prepared, expressed, generated, or isolated using recombinant DNA technology.

[0072] As used herein, the term "antibody" refers to a protein capable of recognizing and specifically binding to an antigen. Common or conventional mammalian antibodies comprise tetramers, which typically consist of two pairs of identical polypeptide chains, each pair consisting of a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50 kDa–70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide with a sufficient sequence of variable domains to confer specificity against a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. If the antibody is derived from a non-human source, the variable domains may undergo further protein engineering to humanize the framework region. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, full-length heavy chain immunoglobulin polypeptides include a variable domain (V... H ) and three constant structural domains (C H1 C H2 and C H3 ) and C H1 and C H2 The hinge area between, where V H The domain is located at the amino terminus of the polypeptide and C H3 The domain is at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide includes a variable domain (V). L ) and constant structural domain (C L ), where V L The domain is located at the amino terminus of the polypeptide and C L The domain is at the carboxyl terminus.

[0073] Within both the full-length light and heavy chains, variable and constant domains are typically linked by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair typically form antigen-binding sites. The variable domains of naturally occurring antibodies generally exhibit the same general structure as a relatively conserved backbone region (FR) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned along the backbone region, enabling binding to specific epitopes. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0074] "Its antigen-binding fragment" refers to the minimum portion of an antibody capable of binding to a specified antigen targeted by the antibody, such as, in the context of typical antibodies produced by B cells, the heavy chain (V... H ) Variable structural domains and light chains (V L At least some complementarity-determining regions (CDRs) of the variable structural domain. The antibody or its antigen-binding fragment can be or may be derived from polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), containing a single V L Domain or V H A domain or a segment combined with a portion of a relative domain (e.g., a whole V with one, two, or three CDRs). L Domains and Partial V H (domains) and fragments generated from Fab expression libraries. scFv molecules are known in the art and described, for example, in U.S. Patent No. 5,892,019.

[0075] As used herein, the term "natural Fc" refers to a molecule containing a sequence of a non-antigen-binding fragment produced by the digestion of an antibody or otherwise, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of natural Fc is preferably human, but can be any type of immunoglobulin. Natural Fc molecules consist of monomeric polypeptides that can be covalently (i.e., disulfide bonds) and non-covalently associated to form dimers or multimers. The number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, and IgG4). An example of natural Fc is a disulfide-bound dimer produced by the digestion of IgG with papain. As used herein, the term "natural Fc" is a generic name for monomeric, dimeric, and multimeric forms.

[0076] As used herein, the term "Fc variant" refers to a molecule or sequence modified from natural Fc but still including a binding site for the rescue receptor FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interactions with the rescue receptor, are known in the art. Thus, the term "Fc variant" can include molecules or sequences humanized from non-human natural Fc. Furthermore, natural Fc includes regions that can be removed or mutated to produce Fc variants to alter certain residues that provide structural features or biological activities not required by the binding proteins of this disclosure. Thus, the term "Fc variant" includes molecules or sequences that lack one or more natural Fc sites or residues, or in which one or more Fc sites or residues have been modified, affecting or involving: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5) complement interaction, (6) binding to Fc receptors other than the rescue receptor, or (7) antibody-dependent cytotoxicity (ADCC).

[0077] As used herein, the term "Fc" encompasses both native Fc and Fc variants as defined above. Like Fc variants and native Fc molecules, the term "Fc" includes molecules in monomeric or multimeric form, whether derived from digestion of intact antibodies or otherwise produced.

[0078] The binding proteins covered by this disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules or derived from them.

[0079] As used herein, the terms “mortar,” “grooves,” and “mortar-grooves” refer to previously developed systems that guide the correct pairing of heavy chains from different antibodies. “Mortars” and “grooves” are engineered into the two heavy chains of different antibodies to facilitate correct pairing. The “mortar-grooves” approach is an efficient way to generate bispecific antibodies by driving heterodimerization in the presence of mutations in the CH3 domain of each half-antibody.

[0080] As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be recognized and bound by the antigen-binding moiety of a binding protein of this disclosure. Target antigens can be used in animals to generate antibodies capable of binding to an epitope of that antigen. Target antigens may have one or more epitopes. For each target antigen recognized by the antigen-binding moiety of a binding protein, competition can be made with the intact antibody that recognizes the target antigen.

[0081] As used herein, the term "antigen binding site" refers to a site generated on the surface of a binding protein disclosed herein, wherein an antigen or an epitope on an antigen is bound.

[0082] As used herein, the term "linker" refers to one or more amino acid residues inserted between the domains of the binding protein of this disclosure. For example, linkers can be inserted between domains at the sequence level. The precise location of the domain transition can be determined by locating peptides that do not form secondary structural elements (such as β-sheets or α-helices), as demonstrated by experimental data or assumed by modeling or secondary structure prediction techniques. Depending on the location of the terminating and initiating residues of the chosen protein fusion, a linker may or may not be necessary, as native linkers are typically found between immunoglobulin domains.

[0083] As used herein, the term "polynucleotide" includes both single and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA.

[0084] As used herein, the term "vector" can refer to a nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows the vector to replicate within the host cell, such as an origin of replication. A vector may also include one or more selective marker genes and other genetic elements known in the art. Certain types of vectors contemplated herein may associate with or incorporate into viruses to facilitate cell transformation.

[0085] As used herein, the term "treatment" refers to reducing the pathology of a disease, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or illness.

[0086] As used herein, the term "administration" means the provision, contact, and / or delivery of the binding protein through any suitable route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, or intralesional injection), percutaneous, topical, oral, rectal, vaginal, nasal, ocular, inhalation, and implantation.

[0087] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly mammalian subjects. Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0088] As used herein, the term "effective amount" or "therapeutic effective amount" of a therapeutic substance (such as a binding protein) applied is an amount sufficient to carry out a specific stated or intended purpose (such as treating cancer or cancer therapy). An "effective amount" may be determined empirically in a conventional manner depending on the stated purpose.

[0089] As used herein, the term "pharmaceutical composition" means a compound or composition that, when appropriately administered to a subject, can induce a desired therapeutic effect. In some aspects, this disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the binding protein of this disclosure. As used herein, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refer to one or more formulation materials suitable for achieving or enhancing the delivery of one or more binding proteins of this disclosure.

[0090] In some aspects, the binding proteins disclosed herein can be formulated into pharmaceutical compositions with pharmaceutically acceptable carriers, excipients, or stabilizers. In some aspects, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any or more routes of administration using methods known in the art. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the bioactivity of the active ingredient. Such formulations may conventionally include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also include compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, and salt-forming counterions such as sodium ions. These and additional known drug carriers, excipients, and / or additives suitable for the formulations described herein are known in the art, for example, as in "Remington: The Science & Practice of Pharmacy," 21st edition, Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 60th edition, Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers suitable for the desired or required administration modality, solubility, and / or stability may be selected.

[0091] In some respects, this article presents a binding protein containing two tumor-associated antigen (TAA) binding sites. In some respects, the tumor-associated antigen (TAA) is differentiation cluster 20 (CD20). CD20 is involved in Ca2+... ++ Transmembrane protein involved in channels, B cell activation, and proliferation. CD20 is a membrane-embedded surface molecule that plays a role in B cell development and differentiation into plasma cells. In some respects, the binding protein contains fragments of rituximab (see, for example, U.S. Patent No. 5,736,137). As used herein, CD20 and LM1486 are used interchangeably.

[0092] In some respects, the tumor-associated antigen (TAA) is phosphatidylinositol proteoglycan-3 (GPC3). GPC3 is a heparan sulfate proteoglycan (HSPG) that is highly expressed in hepatocellular carcinoma, where it attracts Wnt proteins to the cell surface and promotes cell proliferation. As used herein, GPC3 and LM1653 are used interchangeably.

[0093] In some respects, tumor-associated antigens (TAAs) are leucine-rich repeat 15 (LRRC15). LRRC15 is a 581-amino acid type I cell membrane protein belonging to the leucine-rich repeat (LRR) superfamily and possesses binding activity to extracellular matrix (ECM) components. LRRC15 is involved in cell-cell / cell-ECM interactions, cell adhesion, wound healing, and osteogenic differentiation. LRRC15 is highly expressed on cancer-associated fibroblasts (CAFs) within the stroma of various epithelial solid tumors and on mesenchymal tumors such as sarcoma, glioblastoma, and melanoma. + CAF features have shown an association with resistance to PDx therapy across a variety of tumor indications, such as metastatic urothelial carcinoma (mUCC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC).

[0094] In some respects, tumor-associated antigens (TAAs) are B-cell maturation antigens (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17). BCMA is a member of the TNF receptor superfamily, preferentially expressed in mature B lymphocytes, and is important for B-cell development, proliferation, and survival. BCMA is associated with the following: hematologic malignancies, including but not limited to multiple myeloma; and autoimmune / inflammatory diseases, including but not limited to scleroderma, systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, or Sjögren's syndrome.

[0095] In some respects, tumor-associated antigens (TAAs) include prostate six-transmembrane epithelial antigen 2 (STEAP2). STEAP2 is a membrane-intercalated heme protein with a transmembrane domain (TMD) containing a heme chelating prosthetic group, or a metalloreductase. STEAP2 is associated with cancer progression by driving cell proliferation, migration, and invasion. It also affects the transcriptional profile of genes as part of the metastatic cascade. STEAP2 is associated with cancers including, but not limited to, prostate cancer and Ewing sarcoma.

[0096] In some aspects, this document provides a binding protein comprising a T-cell receptor (TCR) binding site. The TCR comprises a heterodimer including highly variable α and β chains. The multicomponent complex of the TCR contains a CD3 co-receptor that plays an important role in activated T cells. In one aspect, a TCR binding protein is provided comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) comprising the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 40, and SEQ ID NO: 41, respectively. In one embodiment, the TCR binding protein comprises V according to SEQ ID NO: 43 or SEQ ID NO: 45. HH On the other hand, the TCR-binding protein contains V according to SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48. HH chain.

[0097] In some respects, this article provides a binding protein comprising a T-cell costimulatory molecule binding site. The costimulatory molecule contains a costimulatory domain capable of enhancing or modulating the response of immune effector cells. The costimulatory domain may include sequences, for example, from one or more of CD3ζ (or CD3z), CD28, CD137(4-1BB), OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. In some respects, the T-cell costimulatory molecule is CD8. In some respects, the T-cell costimulatory molecule is CD137(4-1BB).

[0098] In one non-limiting embodiment, the binding protein activates T cells only when it binds to one or both of the tumor-associated antigen binding sites.

[0099] Some aspects described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen (TAA) binding sites, a T-cell receptor binding site, and a T-cell co-stimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the following formula: V L -C L Furthermore, the third polypeptide chain has a structure represented by the following formula: V H1 -C H1- V HHa -Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -V HHb -Fc bThe first and third polypeptides form the first TAA binding site of the two TAA binding sites, and the second and fourth polypeptides form the second TAA binding site of the two TAA binding sites, wherein: V L It is the variable domain of the immunoglobulin light chain, and V H1 These are the variable domains of the immunoglobulin heavy chain, which together form the TAA-binding domain that specifically binds to tumor-associated antigens; C L It is the constant domain of the immunoglobulin light chain; C H1 It is the CH1 heavy chain constant domain of immunoglobulin; V HHa It is a single-stranded variable domain that specifically binds to the T-cell receptor; V HHb It is a single-stranded variable domain that specifically binds to T cell co-stimulatory molecules; Fc a It is C H2a and C H3a Immunoglobulin heavy chain constant domain; and Fc b It is C H2b and C H3b Immunoglobulin heavy chain constant domain.

[0100] In some aspects, the heavy chain variable domain that specifically binds to T cell co-stimulatory molecules is an immunoglobulin heavy chain variable domain. In some aspects, the heavy chain variable domain that specifically binds to T cell co-stimulatory molecules is a single-domain sequence. In a particular aspect, the heavy chain variable domain that specifically binds to T cell co-stimulatory molecules is a nanobody. In a particular aspect, the heavy chain variable domain that specifically binds to T cell co-stimulatory molecules is derived from camels. In a particular aspect, the heavy chain variable domain that specifically binds to T cell co-stimulatory molecules is a single-domain variable neoantigen receptor. In various non-limiting embodiments, V HHb It comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), which respectively contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; or respectively contain the amino acid sequences of SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171. In various other non-limiting embodiments, V HHb It contains an amino acid sequence that is 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 SEQ ID NO: 52 or 168, or 100% identical to SEQ ID NO: 52 or 168.

[0101] In some respects, V HHbSEQ ID NO: 52 is further modified to further enhance manufacturability. In some respects, V HHb It is SEQ ID NO: 52, but also contains a D30R substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains D30P substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains S75A substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains Y102I substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains S100G substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains L101A substitution. In some respects, V HHb It is SEQ ID NO:52, but also contains Q106N substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains D31S substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains S100P substitution. In some respects, V HHb It is SEQ ID NO: 52, but also contains R52T substitution. In some aspects, the sequence contains one or more substitutions selected from D30R, D30P, S75A, Y102I, S100G, L101A, Q106N, D31S, S100P, or R52T. In some aspects, V HHB Selected from SEQ ID NO: 81-89.

[0102] In some aspects, the heavy chain variable domain that specifically binds to the T cell receptor binding site is an immunoglobulin heavy chain variable domain. In some aspects, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a single-domain sequence. In a particular aspect, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a nanobody. In a particular aspect, the heavy chain variable domain that specifically binds to the T cell receptor binding site is derived from camels. In a particular aspect, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a single-domain variable neoantigen receptor. In various non-limiting embodiments, V HHaIt comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), which respectively contain the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41; or respectively contain the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 40, and SEQ ID NO: 41; or respectively contain the amino acid sequences of SEQ ID NO: 173, SEQ ID NO: 174, and SEQ ID NO: 175. In various other non-limiting embodiments, V HHa It contains an amino acid sequence that is 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 any of SEQ ID NO: 42-48 and 172, or any of SEQ ID NO: 42-48 and 172.

[0103] In some respects, the Fc binding protein a and / or Fc b Derived from IgG antibodies, such as IgG1, IgG2, IgG3, and IgG4. In one non-limiting embodiment, Fc a and / or Fc b Derived from IgG1 antibody.

[0104] In some respects, C H3a and C H3b All are CH3 immunoglobulin heavy chain constant domains. In some respects, Fc a and Fc b C in each of them H3a and C H3b Includes promoting Fc a and Fc b The modification involves heterodimerization. In various non-limiting embodiments, the modification is a substitution to [the molecule] in the Fc [cell]. a and Fc b One of them produces a pestle, and in Fc a and Fc b The other component produces mordant. In one non-limiting embodiment, the pestle is substituted with tryptophan at position 366, and the mordant is substituted with one or more of the following: i) valine at position 407; ii) serine at position 366; and iii) alanine at position 368. In another non-limiting embodiment, the Fc containing the pestle... a or Fc b It also contains cysteine ​​at position 354 and / or contains acetyl phosphate Fc. aor Fc b Cysteine ​​is contained at position 349, and the numbering is based on the Eu index.

[0105] In some respects, C H2a and C H2b Both are CH2 immunoglobulin heavy chain constant domains. In some respects, C H2a and C H2b The immunoglobulin heavy chain constant domains each contain the following substitutions: E233P / L234V / L235A / G236del / S267K, and the numbers are based on the Eu index. In other respects, C... H3a Immunoglobulin heavy chain constant domain or C H3b The immunoglobulin heavy chain constant domain contains H435R and Y436F substitutions, which are numbered according to the Eu index.

[0106] In various non-restrictive implementations, Fc a Contains the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57. In various other non-limiting embodiments, Fc b The amino acid sequence containing SEQ ID NO: 58.

[0107] In some respects, the fourth polypeptide chain contains SEQ ID NO: 61, and the third polypeptide chain contains SEQ ID NO: 59 or SEQ ID NO: 60.

[0108] In some respects, binding proteins contain linkers. The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element to be achieved. For example, glycine, serine, and alanine are optimal for linkers with the greatest flexibility. If a more rigid and extended linker is required, a certain combination of glycine, proline, threonine, and serine is useful. Any combination of amino acid residues with one or more other amino acid residues can be considered a linker to construct a larger peptide linker as needed, depending on the desired properties; the other amino acid residues may be the same as or different from the first amino acid residue. In some respects described herein, binding proteins may also contain: L1, i.e., C located on the third polypeptide chain. H1 With V HHa The linker between; and L2, which is the V located on the third polypeptide chain. HHa With Fc a The linkers between L1 and L2 are either independent linkers or absent. In some aspects described herein, the binding protein may also include: L3, namely C, located on the fourth polypeptide chain. H1 With V HHb The linker between; and L4, which is the V located on the fourth polypeptide chain.HHb With Fc b The linkers between L1, L2, L3, and / or L4 are independently linkers or are absent. In various non-limiting embodiments, L1, L2, L3, and / or L4 comprise one or more repeats of the amino acid sequence of SEQ ID NO: 37 and / or SEQ ID NO: 38.

[0109] In some aspects described herein, the binding protein includes: H1, namely C located on the third polypeptide chain. H1 With V HHa The immunoglobulin hinge region between; and H2, which is V located on the third polypeptide chain. HHa With Fc a The immunoglobulin hinge region between H1 and H2, wherein H1 and H2 are each independently an immunoglobulin hinge region or are absent. In various non-limiting embodiments, H1 comprises SEQ ID NO: 53, DK (SEQ ID NO: 54), SEQ ID NO: 90 or is absent, and H2 comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 90 or is absent.

[0110] In some aspects described herein, the binding protein includes: H3, namely C located on the fourth polypeptide chain. H1 With V HHb The immunoglobulin hinge region between; and H4, which is V located on the fourth polypeptide chain. HHb With Fc b The immunoglobulin hinge region between H3 and H4, wherein H3 and H4 are each independently an immunoglobulin hinge region or are absent. In various non-limiting embodiments, H3 comprises SEQ ID NO: 53, DK (SEQ ID NO: 54), SEQ ID NO: 90 or is absent, and H4 comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 90 or is absent. In various non-limiting embodiments, H3 comprises SEQ ID NO: 53, DK (SEQ ID NO: 54), SEQ ID NO: 90 or is absent, and H4 comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 91 or is absent.

[0111] Some aspects described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T-cell receptor binding site, and a T-cell co-stimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the following formula: V L -CL The third polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H1-L1-V HHa -H2-L2-Fc or V H1 -C H1 -H1-L1-V HHa -L2-H2-Fc, and the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H3-L3-V HHb -H4-L4-Fc b or V H1 -C H1 -H3-L3-V HHb -L4-H4-Fc b L1 is a C-type peptide located on the third polypeptide chain. H1 With V HHa The linker between them, and L2 is a V located on the third polypeptide chain. HHa The linker between L1 and L2 is either an independent linker or absent, and L3 is a C-linker located on the fourth polypeptide chain. H1 With V HHb The linker between them, and L4 is a V located on the fourth polypeptide chain. HHb With Fc b The linkers between, where 3 and L4 are each independently linkers or are absent; where H1 is a C-type linker located on the third polypeptide chain. H1 With V HH The immunoglobulin hinge region between them, and H2 is located on the third polypeptide chain V. HHa With Fc a The immunoglobulin hinge region between, where H1 and H2 are each independently an immunoglobulin hinge region or absent; where H3 is a C located on the fourth polypeptide chain. H1 With V HHb The immunoglobulin hinge region between them, and H4 is a V located on the fourth polypeptide chain. HHb With Fc b The immunoglobulin hinge regions between H3 and H4, where H3 and H4 are either independently immunoglobulin hinge regions or do not exist.

[0112] In some respects, the binding protein contains a tumor-associated antigen (TAA), which is CD20, phosphatidylinositol proteoglycan-3 (GPC3), or a leucine-rich repeat sequence 15 (LRRC15).

[0113] In some respects, tumor-associated antigens (TAAs) are B-cell maturation antigen (BCMA) or prostatic six-transmembrane epithelial antigen 2 (STEAP2).

[0114] In some respects, the TAA-binding domain binds to CD20 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), which respectively contain the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In other respects, the TAA-binding domain binds to CD20 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 182, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 7 and SEQ ID NO: 8, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 7 H and according to SEQ ID NO: 8 V L .

[0115] In some aspects, the TAA-binding domain binds to GPC3 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 19 and SEQ ID NO: 20, respectively. H1 Domain and VL A structural domain; or containing V according to SEQ ID NO:19 H and according to SEQ ID NO: 20 V L .

[0116] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 31 and SEQ ID NO: 32, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 31 H and V according to SEQ ID NO: 32 L .

[0117] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 141 and SEQ ID NO: 142, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 141 H Chain and V according to SEQ ID NO: 142 L .

[0118] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 135, SEQ ID NO: 147, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 148 and SEQ ID NO: 142, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 148 H Chain and V according to SEQ ID NO: 142 L .

[0119] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 135, SEQ ID NO: 152, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 153 and SEQ ID NO: 142, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 153. H Chain and V according to SEQ ID NO: 142 L .

[0120] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 135, SEQ ID NO: 157, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 158, and SEQ ID NO: 140, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 159 and SEQ ID NO: 160, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 159 H Chain and V according to SEQ ID NO: 160 L .

[0121] In some aspects, the TAA-binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 135, SEQ ID NO: 157, SEQ ID NO: 138, SEQ ID NO: 165, and SEQ ID NO: 140, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 159 and SEQ ID NO: 166, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 159 H Chain and V according to SEQ ID NO: 166 L .

[0122] In some aspects, the TAA-binding domain binds to BCMA and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 SEQ ID NO: 117 and SEQ ID NO: 118, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 117. H and according to SEQ ID NO: 118 V L .

[0123] In some aspects, the TAA-binding domain binds to STEAP-2 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) containing the amino acid sequences of SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, and SEQ ID NO: 128, respectively. In various non-limiting embodiments, the TAA-binding domain comprises V that is 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 those in SEQ ID NO: 129 and SEQ ID NO: 130, respectively. H1 Domain and V L A structural domain; or containing V according to SEQ ID NO: 129 H Chain and V according to SEQ ID NO: 130 L .

[0124] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 12.

[0125] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24.

[0126] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36.

[0127] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 119, SEQ ID NO: 121, and SEQ ID NO: 122.

[0128] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 131, SEQ ID NO: 134 and SEQ ID NO: 133.

[0129] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 143, SEQ ID NO: 145 and SEQ ID NO: 146.

[0130] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 143, SEQ ID NO: 150, and SEQ ID NO: 151.

[0131] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 143, SEQ ID NO: 155, and SEQ ID NO: 156.

[0132] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 161, SEQ ID NO: 163, and SEQ ID NO: 164.

[0133] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 167, SEQ ID NO: 163, and SEQ ID NO: 164.

[0134] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 161, SEQ ID NO: 177 and SEQ ID NO: 164.

[0135] Some aspects described herein provide a binding protein comprising the amino acid sequences of SEQ ID NO: 143, SEQ ID NO: 176 and SEQ ID NO: 156.

[0136] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 9, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 11, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 12.

[0137] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 21, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 23, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 24.

[0138] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 33, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 35, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 36.

[0139] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 119, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 121, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 122.

[0140] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 131, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 134, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 133.

[0141] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 143, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 145, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 146.

[0142] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 143, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 150, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 151.

[0143] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 143, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 155, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 156.

[0144] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 161, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 163, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 164.

[0145] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 167, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 163, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 164.

[0146] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 161, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 177, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 164.

[0147] In some respects, the first and second polypeptide chains contain the amino acid sequence of SEQ ID NO: 143, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 176, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 156.

[0148] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:31. H Chain and V according to SEQ ID NO: 32 L In various other non-limiting embodiments, the binding protein comprises the LC chain according to SEQ ID NO: 33 and the V chain according to SEQ ID NO: 34. H-CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 36 and / or SEQ ID NO: 35.

[0149] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:141. H Chain and V according to SEQ ID NO: 142 L In various other non-limiting embodiments, the binding protein comprises LC according to SEQ ID NO: 143 and V according to SEQ ID NO: 144. H -CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 146 and / or SEQ ID NO: 145.

[0150] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:148. H Chain and V according to SEQ ID NO: 142 L In various other non-limiting embodiments, the binding protein comprises LC according to SEQ ID NO: 143 and V according to SEQ ID NO: 149. H -CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 151 and / or SEQ ID NO: 150.

[0151] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:135, SEQ ID NO:152, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:153. H Chain and V according to SEQ ID NO: 142 L In various other non-limiting embodiments, the binding protein comprises LC according to SEQ ID NO: 143 and V according to SEQ ID NO: 154. H -CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 156 and / or SEQ ID NO: 155. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 156 and / or SEQ ID NO: 176.

[0152] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:135, SEQ ID NO:157, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:158, and SEQ ID NO:140, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:159. H Chain and V according to SEQ ID NO: 160 L In various other non-limiting embodiments, the binding protein comprises LC according to SEQ ID NO: 161 and V according to SEQ ID NO: 162. H -CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 164 and / or SEQ ID NO: 163.

[0153] Some aspects described herein provide a binding protein comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:135, SEQ ID NO:157, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:165, and SEQ ID NO:140, respectively. In various non-limiting embodiments, the binding protein comprises V according to SEQ ID NO:159. H Chain and V according to SEQ ID NO: 166 L In various other non-limiting embodiments, the binding protein comprises LC according to SEQ ID NO: 167 and V according to SEQ ID NO: 162. H -CH1. In various other non-limiting embodiments, the binding protein comprises a polypeptide chain according to SEQ ID NO: 164 and / or SEQ ID NO: 163.

[0154] In certain respects, this document provides amino acid sequences with conserved substitutions, wherein up to 10, up to 8, up to 5, or up to 3 amino acids are substituted with amino acids having similar or analogous properties compared to the amino acid sequences disclosed herein.

[0155] In some respects, light chains (V L -C L ) and heavy chain V H and C H1 Associating to form an "antigen-binding arm," and the variable domains within the antigen-binding arm interact to form an "antigen-binding site." As described herein, "antigen-binding site" and "antigen-binding domain" are used interchangeably.

[0156] λ charge pairs The terms “charge pair” and “charge mutation” are used interchangeably throughout the specification and refer to a positively charged amino acid residue and a negatively charged amino acid residue, one located in the light chain region (e.g., the constant light chain region) of the antigen-binding arm and the other located in the heavy chain region (e.g., the constant heavy chain region 1 (C)). H1 In the light chain, these are located at positions designed to promote association between the light and heavy chains. A "λ charge pair" refers to a charge pair in which the positively or negatively charged amino acid residues are located in the λ light chain. A "κ charge pair" refers to a charge pair in which the positively or negatively charged amino acid residues are located in the κ light chain.

[0157] To avoid being bound by theory, it is believed that amino acid residues with opposite charges in the charge pair increase the attraction of the heavy chain to the light chain in the antigen-binding arm, thereby promoting the formation of an antigen-binding arm with the correct heavy and light chains.

[0158] At least one amino acid residue in the charge pair has been engineered into the antigen-binding arm (i.e., at least one amino acid residue in the pair is not a wild-type amino acid residue). In some aspects, both amino acid residues in the charge pair have been engineered into the antigen-binding arm (i.e., neither amino acid residue in the pair is a wild-type amino acid residue).

[0159] The amino acid residues in the charge pairs are typically naturally occurring. Naturally occurring positively charged amino acid residues according to this disclosure include arginine, lysine, and histidine. Naturally occurring negatively charged amino acid residues according to this disclosure include glutamic acid, serine, threonine, and aspartic acid. Although serine and threonine are often described in the art as “uncharged,” they have isoelectric points below 6 and are therefore partially negatively charged at neutral pH. For the purposes of the charge pairs disclosed herein, serine and threonine are examples of negatively charged amino acid residues (along with glutamic acid and aspartic acid).

[0160] Therefore, a charge pair may contain a positively charged amino acid residue selected from arginine, lysine, or histidine at one position in the charge pair and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine at the other position in the charge pair. For example, a charge pair may contain any one of the following amino acid residue pairs: Arginine and aspartic acid; arginine and glutamic acid; arginine and serine; arginine and threonine; lysine and aspartic acid; lysine and glutamic acid; lysine and serine; lysine and threonine; histidine and aspartic acid; histidine and glutamic acid; histidine and serine; and histidine and threonine.

[0161] In some respects, the positively charged amino acid residues in the charge pair are located on the light chain, and the negatively charged amino acid residues in the charge pair are located on the heavy chain. In other respects, the negatively charged amino acid residues are located on the light chain, and the positively charged amino acid residues in the charge pair are located on the heavy chain.

[0162] The binding proteins described herein may contain λ charge pairs in one of the antigen-binding arms. As illustrated herein, λ charge pairs may be introduced at several sites to improve the correct pairing of light and heavy chains in the antigen-binding arm.

[0163] In some respects, λ charge pairs are contained in positively or negatively charged amino acid residues at positions 117, 119, 134, 136, or 178 of the constant light chain λ region (CLλ). In some respects, λ charge pairs are located at C... H1 Positions 141, 185, 128, 145, 183, 185, 173, or 187 contain positively or negatively charged amino acid residues. Elsewhere, the numbering is based on the Eu number. Positions 117, 119, 134, 136, and 178 of CLλ according to the Eu number correspond to amino acid positions 10, 12, 27, 29, and 71 of SEQ ID NO: 98 and 99. C according to the Eu number... H1 Positions 141, 185, 128, 145, 183, 185, 173, and 187 correspond to amino acid positions 24, 68, 11, 28, 66, 68, 56, and 70 of SEQ ID NO: 95-99.

[0164] In some respects, the λ charge pairs are located at one or more of the following position pairs: (i) position 117 in CLλ and position 141 in CH1; (ii) position 117 in CLλ and position 185 in CH1; (iii) position 119 in CLλ and position 128 in CH1; (iv) position 134 in CLλ and position 128 in CH1; (v) position 134 in CLλ and position 145 in CH1; (vi) position 134 in CLλ and position 183 in CH1; (vii) position 136 in CLλ and position 185 in CH1; (viii) position 178 in CLλ and position 173 in CH1; and (ix) position 117 in CLλ and position 187 in CH1.

[0165] In some respects, the λ charge pairs are located at position 117 in CLλ and position 141 in CH1. For example, the λ charge pairs may be selected from the following list: arginine at position 117 in CLλ and aspartic acid at position 141 in CH1; arginine at position 117 in CLλ and glutamic acid at position 141 in CH1; arginine at position 117 in CLλ and serine at position 141 in CH1; arginine at position 117 in CLλ and threonine at position 141 in CH1; lysine at position 117 in CLλ and aspartic acid at position 141 in CH1; lysine at position 117 in CLλ and glutamic acid at position 141 in CH1; lysine at position 117 in CLλ and serine at position 141 in CH1; and lysine at position 117 in CLλ and threonine at position 141 in CH1.

[0166] In some respects, the λ charge pairs are located at position 117 in CLλ and position 185 in CH1. For example, the λ charge pairs may be selected from the following list: arginine at position 117 in CLλ and aspartic acid at position 185 in CH1; arginine at position 117 in CLλ and glutamic acid at position 185 in CH1; arginine at position 117 in CLλ and serine at position 185 in CH1; arginine at position 117 in CLλ and threonine at position 185 in CH1; lysine at position 117 in CLλ and aspartic acid at position 185 in CH1; lysine at position 117 in CLλ and glutamic acid at position 185 in CH1; lysine at position 117 in CLλ and serine at position 185 in CH1; and lysine at position 117 in CLλ and threonine at position 185 in CH1.

[0167] In some respects, the λ charge pairs are located at position 119 in CLλ and position 128 in CH1. For example, the λ charge pairs may be selected from the following list: arginine at position 119 of CLλ and aspartic acid at position 128 of CH1; arginine at position 119 of CLλ and glutamic acid at position 128 of CH1; arginine at position 119 of CLλ and serine at position 128 of CH1; arginine at position 119 of CLλ and threonine at position 128 of CH1; lysine at position 119 of CLλ and aspartic acid at position 128 of CH1; lysine at position 119 of CLλ and glutamic acid at position 128 of CH1; lysine at position 119 of CLλ and serine at position 128 of CH1; and lysine at position 119 of CLλ and threonine at position 128 of CH1.

[0168] In some respects, the λ charge pairs are located at position 134 in CLλ and position 128 in CH1. For example, the λ charge pairs can be selected from the following list: arginine at position 134 in CLλ and aspartic acid at position 128 in CH1; arginine at position 134 in CLλ and glutamic acid at position 128 in CH1; arginine at position 134 in CLλ and serine at position 128 in CH1; arginine at position 134 in CLλ and threonine at position 128 in CH1; lysine at position 134 in CLλ and aspartic acid at position 128 in CH1; lysine at position 134 in CLλ and glutamic acid at position 128 in CH1; lysine at position 134 in CLλ and serine at position 128 in CH1; and lysine at position 134 in CLλ and threonine at position 128 in CH1.

[0169] In some respects, the λ charge pairs are located at position 134 in CLλ and position 145 in CH1. For example, the λ charge pairs can be selected from the following list: arginine at position 134 in CLλ and aspartic acid at position 145 in CH1; arginine at position 134 in CLλ and glutamic acid at position 145 in CH1; arginine at position 134 in CLλ and serine at position 145 in CH1; arginine at position 134 in CLλ and threonine at position 145 in CH1; lysine at position 134 in CLλ and aspartic acid at position 145 in CH1; lysine at position 134 in CLλ and glutamic acid at position 145 in CH1; lysine at position 134 in CLλ and serine at position 145 in CH1; and lysine at position 134 in CLλ and threonine at position 145 in CH1.

[0170] In some respects, the λ charge pairs are located at position 134 in CLλ and position 183 in CH1. For example, the λ charge pairs may be selected from the following list: arginine at position 134 in CLλ and aspartic acid at position 183 in CH1; arginine at position 134 in CLλ and glutamic acid at position 183 in CH1; arginine at position 134 in CLλ and serine at position 183 in CH1; arginine at position 134 in CLλ and threonine at position 183 in CH1; lysine at position 134 in CLλ and aspartic acid at position 183 in CH1; lysine at position 134 in CLλ and glutamic acid at position 183 in CH1; lysine at position 134 in CLλ and serine at position 183 in CH1; and lysine at position 134 in CLλ and threonine at position 183 in CH1.

[0171] In some respects, the λ charge pair is lysine at position 134 of CLλ and either aspartic acid or serine at position 183 of CH1. In the CH1 sequence provided as SEQ ID NO: 97 or SEQ ID NO: 98, EU position 183 is serine, and therefore no modification is needed in CH1 of SEQ ID NO: 100 to create a charge pair with the positively charged amino acid at position 134 of CLλ.

[0172] In some respects, the λ charge pairs are located at position 136 in CLλ and position 185 in CH1. For example, the λ charge pairs may be selected from the following list: arginine at position 136 in CLλ and aspartic acid at position 185 in CH1; arginine at position 136 in CLλ and glutamic acid at position 185 in CH1; arginine at position 136 in CLλ and serine at position 185 in CH1; arginine at position 136 in CLλ and threonine at position 185 in CH1; lysine at position 136 in CLλ and aspartic acid at position 185 in CH1; lysine at position 136 in CLλ and glutamic acid at position 185 in CH1; lysine at position 136 in CLλ and serine at position 185 in CH1; and lysine at position 136 in CLλ and threonine at position 185 in CH1.

[0173] In some respects, the λ charge pairs are located at position 178 in CLλ and position 173 in CH1. For example, the λ charge pairs can be selected from the following list: arginine at position 178 in CLλ and aspartic acid at position 173 in CH1; arginine at position 178 in CLλ and glutamic acid at position 173 in CH1; arginine at position 178 in CLλ and serine at position 173 in CH1; arginine at position 178 in CLλ and threonine at position 173 in CH1; lysine at position 178 in CLλ and aspartic acid at position 173 in CH1; lysine at position 178 in CLλ and glutamic acid at position 173 in CH1; lysine at position 178 in CLλ and serine at position 173 in CH1; and lysine at position 178 in CLλ and threonine at position 173 in CH1.

[0174] In some respects, the first antigen-binding arm contains more than one λ charge pair. For example, the first antigen-binding arm may contain two, three, four, five, six, seven, eight, or nine λ charge pairs at the positions (i) to (ix) above.

[0175] In each respect, the CLλ of the first polypeptide chain and / or the CLλ of the second polypeptide chain each contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:98-99.

[0176] In all other respects, CLλ comprises the amino acid sequence according to SEQ ID NO: 105, and corresponds to C H1 It comprises the amino acid sequence according to SEQ ID NO: 104. In various other respects, CLλ comprises the amino acid sequence according to SEQ ID NO: 105, and corresponds to CH1 It contains the amino acid sequence according to SEQ ID NO: 220.

[0177] In all other respects, CLλ contains the amino acid sequence according to SEQ ID NO: 107, and corresponds to C H1 It contains the amino acid sequence according to SEQ ID NO: 106.

[0178] Engineered disulfide bonds In some respects, in addition to or replacing λ charge pairs, binding proteins also contain engineered disulfide bonds. The term "engineered disulfide bond" refers to a C-type bond in one of the antigen-binding arms. H1 -C L Interface (e.g., C) H1 220 and C L The natural interchain disulfide bond at position 212 has been replaced by an engineered (non-natural) interchain disulfide bond, while another antigen-binding arm at C... H1 -C L The interface contains natural interchain disulfide bonds. Engineered disulfide bonds are typically created by engineering cysteine ​​into the C-terminus of the light chain. L and the corresponding heavy chain C H1 The cysteine ​​residues that normally form interchain disulfide bonds are replaced to form them. Publications relating to the introduction of engineered disulfide bonds into binding proteins to promote heterodimerization can be found, for example, in U.S. Patent Nos. 9,527,927 and Mazor, 2015, the full text of which are incorporated herein by reference.

[0179] The formation of disulfide bonds between cysteine ​​residues occurs during the folding of many proteins entering the secretory pathway. When a polypeptide chain folds, adjacent cysteine ​​residues can form a covalent bond during a process catalyzed by members of the protein disulfide isomerase family. As used herein, the term "disulfide bond linkage" or "disulfide-linked" refers to a single covalent bond formed by the coupling of thiol groups, particularly cysteine ​​residues. In some respects, the covalent bond between two cysteine ​​residues is between the two sulfur atoms of each residue. However, depending on the environment, not all protein species may always have disulfide bonds present, for example, in the case of disulfide bond reduction. Therefore, in some respects, the term "disulfide bond linkage" or "disulfide-linked" (whether natural or engineered) also refers to the presence of two cysteine ​​residues capable of forming a disulfide bond linkage, regardless of whether they are actually linked at that single point in time.

[0180] In some respects of this article, as used herein, “V12 DS” or “V12” refers to the removal of C. H1 The natural interchain disulfide bonds in the / CLλ interface, and subsequently the introduction of substituted disulfide bonds with the following four mutations: C H1F126C / C220V in the structural domain, and the mutation S122C / C212V in the λ-constant structural domain.

[0181] Therefore, in some aspects: (i) in the CL of the first polypeptide chain and the CH1 of the third polypeptide chain, wherein the CL of the first polypeptide chain is CLλ, and wherein an engineered cysteine ​​pair is located at position 122 of the CLλ of the first polypeptide chain and position 126 of the CH1 of the third polypeptide chain, and wherein the CLλ of the first polypeptide chain contains a non-cysteine ​​residue at position 212, and the CH1 of the third polypeptide chain contains a non-cysteine ​​residue at position 220, optionally wherein the non-cysteine ​​residue is valine; or (ii) in the CL of the second polypeptide chain and the CH1 of the fourth polypeptide chain, wherein the CL of the first polypeptide chain is CLλ, and wherein an engineered cysteine ​​pair is located at position 122 of the CLλ of the second polypeptide chain and position 126 of the CH1 of the fourth polypeptide chain, and wherein the CLλ of the second polypeptide chain contains a non-cysteine ​​residue at position 212, and the CH1 of the fourth polypeptide chain contains a non-cysteine ​​residue at position 220, optionally wherein the non-cysteine ​​residue is valine.

[0182] In some respects, the cysteine ​​pair is engineered into: (i) the CL of the first polypeptide chain and the CH1 of the third polypeptide chain, wherein the CL of the first polypeptide chain is CLκ, and wherein the engineered cysteine ​​pair is located at position 121 of the CLκ of the first polypeptide chain and position 126 of the CH1 of the third polypeptide chain, and wherein the CLκ of the first polypeptide chain contains a non-cysteine ​​residue at position 214, and the CH1 of the third polypeptide chain contains a non-cysteine ​​residue at position 220, optionally wherein the non-cysteine ​​residue is valine; or (ii) the CL of the second polypeptide chain and the CH1 of the fourth polypeptide chain, wherein the CL of the first polypeptide chain is CLκ, and wherein the engineered cysteine ​​pair is located at position 121 of the CLκ of the second polypeptide chain and position 126 of the CH1 of the fourth polypeptide chain, and wherein the CLκ of the second polypeptide chain contains a non-cysteine ​​residue at position 214, and the CH1 of the fourth polypeptide chain contains a non-cysteine ​​residue at position 220, optionally wherein the non-cysteine ​​residue is valine.

[0183] An exemplary amino acid sequence of CLλ containing engineered cysteine ​​is provided as SEQ ID NO: 97, and an exemplary amino acid sequence of CH1 containing the corresponding engineered cysteine ​​to form an engineered disulfide bond is provided as SEQ ID NO: 99.

[0184] In the binding proteins illustrated herein, engineered disulfide bonds are present on the “first” antigen-binding arm containing a λ charge pair, and native disulfide bonds are present on the “second” antigen-binding arm not containing a λ charge pair. However, a reverse arrangement is also specifically envisioned, in which native disulfide bonds are present on the first antigen-binding arm, and engineered disulfide bonds are present on the second antigen-binding arm.

[0185] In some respects, cysteine ​​pairs are engineered into the constant light chain κ region (CLκ) and CH1, and are located at position 121 of CLκ and position 126 of CH1, wherein the same CLκ contains a non-cysteine ​​residue at position 214, and the same CH1 contains a non-cysteine ​​residue at position 220. In some respects, the non-cysteine ​​residue is valine.

[0186] κ charge pair In some respects, the antigen-binding arm contains a constant light chain κ region (CLκ). As described herein, techniques such as light chain affinity chromatography utilizing affinity resins specific for CLκ or CLλ can be used for the light chain-selective purification of antibodies. Examples of such affinity resins include LambdaFabSelect and KappaSelect resins from GE Healthcare. Such methods can be used to selectively purify binding proteins containing both CLκ and CLλ, and therefore can be used to improve the production of this form of bispecific antibody.

[0187] An example of the CLκ amino acid sequence is provided as SEQ ID NO: 100.

[0188] In some respects, one of the antigen-binding arms contains a κ charge pair. As described above, a κ charge pair refers to a positively charged amino acid residue and a negatively charged amino acid residue, one of which is located in the κ light chain (e.g., CLκ) of the antigen-binding arm and the other in the heavy chain (e.g., CH1) of the antigen-binding arm, located at positions designed to facilitate association between the light chain of the second antigen-binding arm and CH1.

[0189] In some aspects, the second antigen-binding arm comprises a κ-charge pair located at position 133 in CLκ and position 183 in CH1. In some aspects, the negatively charged amino acid residue in the κ-charge pair is at position 133 in CLκ, and the positively charged amino acid residue in the κ-charge pair is at position 183 in CH1. In other aspects, the positively charged amino acid residue in the κ-charge pair is at position 133 in CLκ, and the negatively charged amino acid residue in the κ-charge pair is at position 183 in CH1. In some aspects, the negatively charged amino acid residue (e.g., at position 133 in CLκ) is glutamic acid, and the positively charged amino acid residue (e.g., at position 183 in CH1) is lysine. Elsewhere, this numbering is based on Eu numbering.

[0190] Position 133 of CLκ according to Eu number corresponds to amino acid position 26 of SEQ ID NO: 100. Position 183 of CH1 according to Eu number corresponds to amino acid position 66 of SEQ ID NO: 95 and 97.

[0191] In some aspects, the binding protein comprises a first antigen-binding arm having a λ charge pair as described above and a second antigen-binding arm having a κ charge pair as described above, wherein the binding protein comprises engineered disulfide bonds. For example, in one aspect, the first antigen-binding arm comprises a λ charge pair (e.g., at position 117 in CLλ and position 141 in CH1), and a disulfide bond between the light chain and CH1 is formed between cysteine ​​pairs engineered into CLλ and CH1; and the second antigen-binding arm comprises a κ charge pair (e.g., at position 133 in CLκ and position 183 in CH1), and a disulfide bond between the light chain and CH1 is formed between native cysteine ​​pairs in CLκ and CH1 of the second light chain.

[0192] On the other hand, the first antigen-binding arm contains a λ charge pair (e.g., at position 117 in CLλ and position 141 in CH1), and a disulfide bond between the first light chain and CH1 is formed between the native cysteine ​​pair in CLλ and CH1 of the light chain; and the second antigen-binding arm contains a κ charge pair (e.g., at position 133 in CLκ and position 183 in CH1), and a disulfide bond between the light chain of the second antigen-binding arm and CH1 is formed between the cysteine ​​pair engineered into the light chain in CLκ and CH1.

[0193] In some respects, the CLκ of the first or second polypeptide chain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 100.

[0194] In all respects, the corresponding C in each of one or more λ charge pairs H1 Contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:95-97, and / or a corresponding C in the κ charge pair. H1 It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 100.

[0195] Therapeutic compositions and methods The binding proteins of this disclosure can be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, mouse, or humanized antibodies. Some aspects of this disclosure relate to isolated nucleic acid molecules encoding binding proteins as described herein. In each aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 184-219. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 184-186. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 187-189. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 190-192. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 193-196. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 196-198.

[0196] In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 199-201. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 202-204. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 205-207. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 208-210. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 211-213. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 214-216. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 217-219.

[0197] The isolated nucleic acid molecules may be contained in a vector. In one aspect, the vector contains isolated nucleic acid molecules of any of SEQ ID NO: 184-219.

[0198] In some aspects, the methods disclosed herein relate to treating a subject's cancer by administering an effective amount of the binding protein. This disclosure also provides a therapeutically effective amount of the binding protein for use in treating a subject's cancer. This disclosure further provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament. This disclosure also provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0199] In the treatment method disclosed herein, the binding protein can preferentially activate a subset of T cells in the subject. This subset of T cells may be CD8+ T cells. CD8+ T cells can be preferentially activated compared to CD4+ T cells. Preferential activation of CD8+ T cells reduces binding to pro-tumor T cells and CD4+ T cells, which produce most of the cytokines that cause release syndrome (CRS). Furthermore, preferential binding of CD8+ T cells induces pyroptosis.

[0200] T cell activation using the methods of this disclosure can be determined by measuring the percentage of surface interleukin-2 receptor α chain positive (CD25+) T cells. The percentage of surface CD25+ T cells, which are CD8 T cells, can be higher than the percentage of surface CD25+ T cells, which are CD4 T cells. In a specific aspect, T cell activation can be determined by the CD69+ / CD25+ T cell ratio. In a specific aspect, T cell activation can be determined by measuring the levels of cytokines released by activated T cells.

[0201] Compared to that produced by bispecific T-cell connectives (BiTEs) previously known in the art, the therapeutic methods of this disclosure can cause reduced binding of regulatory T cells (Tregs), increased cytolytic activity, and / or a reduced incidence of cytokine release syndrome (CRS). This disclosure also provides a therapeutically effective amount of the binding protein for use in reducing regulatory T-cell (Treg) binding, increasing cytolytic activity, and / or reducing the incidence of cytokine release syndrome (CRS) compared to that produced by bispecific T-cell connectives (BiTEs) previously known in the art.

[0202] In some respects, cancer includes B-cell malignancies, liver cancer, hepatocellular carcinoma (HCC), lung cancer, non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer (sqNSCLC), ovarian cancer, clear cell ovarian cancer, carcinoma, Merkel cell carcinoma, gastric cancer, hepatoblastoma, nephroblastoma, melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, thyroid cancer, multiple myeloma, prostate cancer, or Ewing sarcoma.

[0203] In some respects, cancers include B-cell malignancies, liver cancer, hepatocellular carcinoma (HCC), melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, and / or thyroid cancer.

[0204] In some respects, B-cell malignancies, including chronic malignancies, are non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, or primary mediastinal large B-cell lymphoma (PMBCL).

[0205] In some respects, B-cell malignancies, including chronic malignancies, are non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, primary mediastinal large B-cell lymphoma (PMBCL), or small lymphocytic leukemia (SLL).

[0206] In various non-limiting embodiments, CLL, MCL, FL, and / or DLBCL are positive for AA amyloidosis. In various other non-limiting embodiments, CLL, MCL, FL, and / or DLBCL are fibrin-associated (FA).

[0207] In some embodiments, the cancer is a B-cell malignancy. In another embodiment, the cancer is hepatocellular carcinoma. In yet another embodiment, the cancer is multiple myeloma. In yet another embodiment, the cancer is osteosarcoma.

[0208] In some embodiments, the tumor-associated antigen is CD20, and the cancer is a B-cell malignancy. In various non-limiting embodiments, the B-cell malignancy is non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, or primary mediastinal large B-cell lymphoma (PMBCL). In another embodiment, the B-cell malignancy is small lymphocytic leukemia (SLL).

[0209] In some respects, the tumor-associated antigen is phosphatidylinositol proteoglycan-3 (GPC3), and the cancer is liver cancer or hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer (sqNSCLC), ovarian cancer, clear cell ovarian cancer, carcinoma, Merkel cell carcinoma, gastric cancer, hepatoblastoma, or nephroblastoma.

[0210] In some respects, tumor-associated antigens are leucine-rich repeat sequences 15 (LRRC15), and the cancers are melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, or thyroid cancer.

[0211] In some respects, the tumor-associated antigen is the B-cell maturation antigen (BCMA), and the cancer is multiple myeloma.

[0212] In some respects, the tumor-associated antigen is STEAP2, and the cancer is prostate cancer or Ewing sarcoma.

[0213] In some aspects, the methods disclosed herein relate to treating an inflammatory disease in a subject of need by administering an effective amount of the binding protein. This disclosure also provides a therapeutically effective amount of the binding protein for use in treating an inflammatory disease in a subject. This disclosure further provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament. This disclosure also provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament for treating inflammatory diseases.

[0214] In some aspects, the methods disclosed herein relate to treating inflammatory diseases and / or autoimmune disorders in subjects of need by administering an effective amount of the binding protein. This disclosure also provides a therapeutically effective amount of the binding protein for use in treating inflammatory diseases and / or autoimmune disorders in subjects. This disclosure further provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament. This disclosure also provides the use of the binding protein and / or pharmaceutical composition in the manufacture of a medicament for treating inflammatory diseases and / or autoimmune disorders.

[0215] In some aspects, the inflammatory disease and / or autoimmune condition is, but is not limited to, systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), Sjögren's syndrome, or antineutrophil cytoplasmic autoantibody (ANCA) vasculitis. In one embodiment, the inflammatory disease and / or autoimmune condition is systemic lupus erythematosus (SLE). In another embodiment, the inflammatory disease and / or autoimmune condition is myositis. In another embodiment, the inflammatory disease and / or autoimmune condition is rheumatoid arthritis (RA). In another embodiment, the inflammatory disease and / or autoimmune condition is Sjögren's syndrome. In another embodiment, the inflammatory disease and / or autoimmune condition is antineutrophil cytoplasmic autoantibody (ANCA) vasculitis.

[0216] In some aspects, the methods disclosed herein relate to treating a subject with inflammatory diseases and / or autoimmune disorders involving B cells by administering an effective amount of the binding protein. This disclosure also provides a therapeutically effective amount of the binding protein for use in treating a subject with inflammatory diseases involving B cells.

[0217] In some respects, the tumor-associated antigen is CD20, and inflammatory diseases or autoimmune disorders involve B cells.

[0218] In some respects, the tumor-associated antigen is the B-cell maturation antigen (BCMA), and the autoimmune disease is scleroderma, systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), or Sjögren's syndrome.

[0219] In all respects, the conjugate protein, composition, pharmaceutical composition and / or drug may be administered via any suitable route of administration, including but not limited to oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional or intracranial injection), percutaneous, local, oral, rectal, vaginal, nasal, ocular, inhalation and implantation.

[0220] Claims or descriptions including "or" among one or more members of a group are considered to satisfy the following conditions: one member of the group, more than one member of the group, or all members of the group are present in, used in, or otherwise associated with the given product or process, unless indicated to the contrary or obvious from the context. This disclosure includes an aspect where exactly one member of the group is present in, used in, or otherwise associated with the given product or process. This disclosure includes an aspect where more than one member of the group, or all members of the group, are present in, used in, or otherwise associated with the given product or process.

[0221] Furthermore, this disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. When elements are presented as a list, for example in Markush group format, each subgroup of elements is also disclosed, and any element may be removed from the group.

[0222] It should be understood that, generally speaking, where an aspect of this disclosure is referred to as including specific elements and / or features, certain aspects of this disclosure are composed of or substantially composed of such elements and / or features. For simplicity, those aspects are not specifically described in these words herein.

[0223] In view of this disclosure, the binding proteins, pharmaceutical compositions, nucleic acid molecules, carriers, methods, compositions and pharmaceuticals for use described herein can be configured by those skilled in the art to meet desired needs.

[0224] Without limiting this disclosure, many embodiments of this disclosure are described herein for illustrative purposes.

[0225] Implementation Scheme 1. A binding protein comprising four polypeptide chains forming two tumor-associated antigen (TAA) binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the first polypeptide chain and the second polypeptide chain have a structure represented by the following formula: V L -C L The third polypeptide chain has a structure represented by the following formula: V H1 -C H1- V HHa -Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -V HHb -Fc b Wherein the first polypeptide and the third polypeptide form the first TAA binding site of the two TAA binding sites, and the second polypeptide and the fourth polypeptide form the second TAA binding site of the two TAA binding sites, and wherein: V L It is the variable domain of the immunoglobulin light chain, and V H1 These are the variable domains of the immunoglobulin heavy chain, which together form the TAA-binding domain that specifically binds to tumor-associated antigens; C L It is the constant domain of the immunoglobulin light chain; C H1It is the CH1 heavy chain constant domain of immunoglobulin; V HHa It is a single-stranded variable domain that specifically binds to the T-cell receptor; V HHb It is a single-stranded variable domain that specifically binds to T cell co-stimulatory molecules; Fc a It is C H2a and C H3a Immunoglobulin heavy chain constant domain; and Fc b It is C H2b and C H3b Immunoglobulin heavy chain constant domain.

[0226] Implementation Scheme 2. The binding protein according to Implementation Scheme 1, wherein the binding protein further comprises: L1, namely, C located on the third polypeptide chain. H1 With V HHa The linker between; and L2, namely V located on the third polypeptide chain. HHa With the Fc a The joints between L1 and L2, where L1 and L2 are either joints or do not exist independently.

[0227] Implementation Scheme 3. The binding protein according to Implementation Scheme 1 or Implementation Scheme 2, wherein the binding protein further comprises: L3, namely, C located on the fourth polypeptide chain. H1 With V HHb The linker between; and L4, namely the V located on the fourth polypeptide chain. HHb With Fc b The joints between L3 and L4 are either independent joints or do not exist.

[0228] Implementation Scheme 4. The binding protein according to any one of Implementation Schemes 1 to 3, wherein the binding protein further comprises: H1, namely, C located on the third polypeptide chain. H1 With V HHa The immunoglobulin hinge region between; and H2, i.e., V located on the third polypeptide chain. HHa With the Fc a The immunoglobulin hinge regions between H1 and H2, where H1 and H2 are either independently immunoglobulin hinge regions or do not exist.

[0229] Implementation Scheme 5. The binding protein according to Implementation Scheme 4, wherein H1 contains SEQ ID NO: 53 or DK (SEQ ID NO: 54) or SEQ ID NO: 90 or is not present, and wherein H2 contains SEQ ID NO: 53 or SEQ ID NO: 55 or SEQ ID NO: 90 or is not present.

[0230] Implementation Scheme 6. The binding protein according to any one of Implementation Schemes 1 to 5, wherein the binding protein further comprises: H3, namely, C located on the fourth polypeptide chain. H1 With V HHb The immunoglobulin hinge region between; and H4, namely V located on the fourth polypeptide chain. HHb With the Fc b The immunoglobulin hinge regions between H3 and H4, where H3 and H4 are either independently immunoglobulin hinge regions or do not exist.

[0231] Implementation Scheme 7. The binding protein according to Implementation Scheme 6, wherein H3 contains SEQ ID NO: 53 or DK (SEQ ID NO: 54) or SEQ ID NO: 90 or is not present, and wherein H4 contains SEQ ID NO: 53 or SEQ ID NO: 55 or SEQ ID NO: 90 or is not present.

[0232] Implementation Scheme 8. The binding protein according to any one of Implementation Schemes 1 to 7, wherein the first polypeptide chain and the second polypeptide chain have a structure represented by the following formula: V L -C L The third polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H1-L1-V HHa -H2-L2-Fc a or V H1 -C H1 -H1-L1-V HHa -L2-H2-Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H3-L3-V HHb -H4-L4-Fc b or V H1 -C H1 -H3-L3-V HHb -L4-H4-Fc b .

[0233] Implementation Scheme 9. The binding protein according to any one of Implementation Schemes 1 to 8, wherein Fc a and / or Fc b From IgG antibodies.

[0234] Implementation Scheme 10. The binding protein according to Implementation Scheme 9, wherein the Fc a And / or the Fc b Derived from IgG1 antibody.

[0235] Implementation Scheme 11. The binding protein according to any one of Implementation Schemes 1 to 10, wherein the binding protein activates T cells only when it binds to a tumor-associated antigen at one or both of the antigen binding sites.

[0236] Implementation Scheme 12. The binding protein according to any one of Implementation Schemes 2 to 11, wherein L1, L2, L3 and / or L4 comprises one or more repeats of the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38 and / or SEQ ID NO: 111.

[0237] Implementation Scheme 13. The binding protein according to any one of Implementation Schemes 1 to 12, wherein the V HHa The heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) contain the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively.

[0238] Implementation Scheme 14. The binding protein according to any one of Implementation Schemes 1 to 12, wherein the V HHa The heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) contain the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 40, and SEQ ID NO: 41, respectively.

[0239] Implementation Scheme 15. The binding protein according to any one of Implementation Schemes 1 to 14, wherein the V HHa Contains 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% of the same amino acid sequence as any of SEQ ID NO: 42-48, or any of SEQ ID NO: 42-48.

[0240] Implementation Scheme 16. The binding protein according to any one of Implementation Schemes 1 to 15, wherein the T cell co-stimulatory molecule is CD8.

[0241] Implementation Scheme 17. The binding protein according to any one of Implementation Schemes 1 to 16, wherein the V HHb Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) contain the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively.

[0242] Implementation Scheme 18. The binding protein according to any one of Implementation Schemes 1 to 17, wherein the V HHb Contains an amino acid sequence that is 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 SEQ ID NO: 52, or SEQ ID NO: 52.

[0243] Implementation Scheme 19. The binding protein according to any one of Implementation Schemes 1 to 18, wherein Fc a and Fc b C in each of them H3a and C H3b Includes promoting Fc a and Fc b The modification of heterodimerization.

[0244] Implementation Scheme 20. The binding protein according to Implementation Scheme 19, wherein the modification is in Fc a and Fc b One of them produces a pestle and in Fc a and Fc b The substitution of mordant is produced in another of the mordants, wherein the mordant is substituted with tryptophan at position 366, and wherein the mordant is substituted with one or more of the following: (i) substituted with valine at position 407; (ii) substituted with serine at position 366; and (iii) substituted with alanine at position 368.

[0245] Implementation Scheme 21. The Fc containing the pestle of the binding protein according to Implementation Scheme 20. a or the Fc b It also contains cysteine ​​at position 354, and / or contains the Fc of the mordant. a or the Fc b Cysteine ​​is present at position 349.

[0246] Implementation Scheme 22. The binding protein according to any one of Implementation Schemes 1 to 21, wherein the C H2a and C H2b Each of the constant domains of the immunoglobulin heavy chain contains the following substitutions: E233P / L234V / L235A / G236del / S267K.

[0247] Implementation Scheme 23. The binding protein according to any one of Implementation Schemes 1 to 22, wherein the C H3a Immunoglobulin heavy chain constant domain or the C H3b The constant domain of the immunoglobulin heavy chain contains H435R and Y436F substitutions.

[0248] Implementation Scheme 24. The binding protein according to any one of Implementation Schemes 1 to 23, wherein Fc a The amino acid sequence containing SEQ ID NO: 56 or SEQ ID NO: 57.

[0249] Implementation Scheme 25. The binding protein according to any one of Implementation Schemes 1 to 24, wherein Fc b The amino acid sequence containing SEQ IDNO: 58.

[0250] Implementation Scheme 26. The binding protein according to any one of Implementation Schemes 1 to 25, wherein the third polypeptide chain comprises SEQ ID NO: 59 or SEQ ID NO: 60, and the fourth polypeptide chain comprises SEQ ID NO: 61.

[0251] Implementation Scheme 27. The binding protein according to any one of Implementation Schemes 1 to 25, wherein the C of the first polypeptide chain L and / or the C of the second polypeptide chain L The binding protein contains a constant light chain λ region (CLλ); and the binding protein contains at least one λ charge pair, wherein the λ charge pair is located at the CLλ of the first polypeptide chain and the Cλ of the third polypeptide chain. H1 The corresponding C H1 Between, or the λ charge pair in the second polypeptide's CLλ and the C of the fourth polypeptide chain. H1 The corresponding C H1 Between, and wherein the λ charge pair is located at one or more of the following positions: (i) position 117 in the CLλ and the corresponding C H1 (ii) Position 141 in the CLλ and the corresponding C H1 (iii) Position 185 in the CLλ and the corresponding C H1 (iv) Position 128 in the CLλ and the corresponding C H1 Position 128 in the middle; (v) Position 134 in the CLλ and the corresponding C H1 Position 145 in the middle; (vi) Position 134 in the CLλ and the corresponding C H1 Position 183 in the middle; (vii) Position 136 in the CLλ and the corresponding C H1 (viii) Position 185 in the CLλ and the corresponding C H1 Position 173 in; and (ix) position 117 in CLλ and the corresponding C H1Position 187; wherein the λ charge pair comprises a positively charged amino acid residue selected from arginine, lysine and histidine located at one of the positions in the λ charge pair and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine and threonine located at the other position in the λ charge pair; and wherein the numbering is based on the EU index.

[0252] Implementation Scheme 28. The binding protein according to Implementation Scheme 27, wherein the λ charge pair is selected from the following list: (a) the arginine at position 117 of CLλ and the corresponding C H1 (a) Aspartic acid at position 141 in the CLλ; (b) arginine at position 117 in the CLλ and the corresponding C H1 (c) Glutamic acid at position 141 in the CLλ; and the corresponding C H1 (d) Serine at position 141 in the CLλ; and the corresponding C H1 (e) the threonine at position 141 in the CLλ; and the lysine at position 117 in the CLλ and the corresponding C H1 (f) Aspartic acid at position 141 in the CLλ; and the corresponding C H1 Glutamic acid at position 141 in the CLλ; (g) lysine at position 117 in the CLλ and the corresponding C H1 The serine at position 141 in (h) and the lysine at position 117 in CLλ and the corresponding C H1 Threonine at position 141.

[0253] Implementation Scheme 29. The binding protein according to Implementation Scheme 27 or Implementation Scheme 28, wherein the CLλ of the first polypeptide chain and / or the CLλ of the second polypeptide chain each comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 98-99.

[0254] Implementation Scheme 30. The binding protein according to any one of Implementation Schemes 1 to 29, wherein the C of the first polypeptide chain L and / or the C of the second polypeptide chain L It includes a constant light chain κ region (CLκ); and a κ charge pair, wherein the κ charge pair is located at CLκ of the first polypeptide chain and C of the third polypeptide chain. H1 The corresponding C H1 Between, or the κ charge pair in the second polypeptide chain CLκ and the C of the fourth polypeptide chainH1 The corresponding C H1 Between; wherein the κ charge pair is located at position 117 in the CLκ and the corresponding C H1 Position 141 of the κ charge pair contains a positively charged amino acid residue selected from arginine, lysine, and histidine at one of the positions in the κ charge pair and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, and threonine at the other position in the κ charge pair.

[0255] Implementation Scheme 31. The binding protein according to Implementation Scheme 30, wherein the negatively charged amino acid residue in the κ charge pair is located at position 133 of the CLκ, and the positively charged amino acid residue is located at the corresponding C H1 At position 183, optionally wherein the negatively charged amino acid residue at position 133 of CLκ is glutamic acid, and optionally wherein the corresponding C H1 The positively charged amino acid residue at position 183 is lysine.

[0256] Implementation Scheme 32. The binding protein according to Implementation Scheme 30 or Implementation Scheme 31, wherein the CLκ of the first polypeptide chain or the second polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 100.

[0257] Implementation Scheme 33. The binding protein according to any one of Implementation Schemes 30 to 32, wherein the corresponding C in each of one or more λ charge pairs H1 Contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 95-97, and / or the corresponding C in the κ charge pair. H1 It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 100.

[0258] Implementation Scheme 34. The binding protein according to any one of Implementation Schemes 27 to 33, wherein the CLλ comprises the amino acid sequence according to SEQ ID NO: 105, and the corresponding C H1 It contains the amino acid sequence according to SEQ ID NO: 104.

[0259] Implementation Scheme 35. The binding protein according to any one of Implementation Schemes 1 to 34, wherein: (i) in the C engineered into the first polypeptide chain L and the C of the third polypeptide chain H1 The C-chain formed between cysteine ​​pairs in the first polypeptide chain L With the C of the third polypeptide chain H1 The disulfide bonds between them, and the C of the second polypeptide chain formed between natural cysteine ​​pairs. L With the C of the fourth polypeptide chain H1 The disulfide bond between; or (ii) in the C-terminal linker engineered into the second polypeptide chain. L and the C of the fourth polypeptide chain H1 The C-chain formed between cysteine ​​pairs in the second polypeptide chain L With the C of the fourth polypeptide chain H1 The disulfide bonds between them, and the C of the first polypeptide chain formed between natural cysteine ​​pairs. L With the C of the third polypeptide chain H1 The disulfide bonds between them are connected.

[0260] Implementation Scheme 36. The binding protein according to Implementation Scheme 35, wherein the cysteine ​​pair is engineered to: (i) the C of the first polypeptide chain L and the C of the third polypeptide chain H1 In, wherein the C of the first polypeptide chain L It is CLλ, and wherein the engineered cysteine ​​pair is located at position 122 of the CLλ in the first polypeptide chain and at position C in the third polypeptide chain. H1 At position 126, and wherein the CLλ of the first polypeptide chain contains a non-cysteine ​​residue at position 212, and the C of the third polypeptide chain... H1 A non-cysteine ​​residue is contained at position 220, optionally wherein said non-cysteine ​​residue is valine; or (ii) the C of the second polypeptide chain. L and the C of the fourth polypeptide chain H1 In, wherein the C of the first polypeptide chain L It is CLλ, and wherein the engineered cysteine ​​pair is located at position 122 of the CLλ in the second polypeptide chain and at position C in the fourth polypeptide chain. H1 At position 126, and wherein the CLλ of the second polypeptide chain contains a non-cysteine ​​residue at position 212, and the C of the fourth polypeptide chain... H1 A noncysteine ​​residue is contained at position 220, wherein the noncysteine ​​residue is optionally valine.

[0261] Implementation Scheme 37. The binding protein according to Implementation Scheme 35, wherein the cysteine ​​pair is engineered to: (i) the C of the first polypeptide chain L and the C of the third polypeptide chain H1 In, wherein the C of the first polypeptide chain L It is CLκ, and wherein the engineered cysteine ​​pair is located at position 121 of the CLκ in the first polypeptide chain and at position C in the third polypeptide chain. H1 At position 126, and wherein the CLκ of the first polypeptide chain contains a non-cysteine ​​residue at position 214, and the C of the third polypeptide chain... H1 A non-cysteine ​​residue is contained at position 220, optionally wherein said non-cysteine ​​residue is valine; or (ii) the C of the second polypeptide chain. L and the C of the fourth polypeptide chain H1 In, wherein the C of the first polypeptide chain L It is CLκ, and wherein the engineered cysteine ​​pair is located at position 121 of the CLκ in the second polypeptide chain and at position C in the fourth polypeptide chain. H1 At position 126, and wherein the CLκ of the second polypeptide chain contains a non-cysteine ​​residue at position 214, and the C of the fourth polypeptide chain... H1 A noncysteine ​​residue is contained at position 220, wherein the noncysteine ​​residue is optionally valine.

[0262] Implementation Scheme 38. The binding protein according to any one of Implementation Schemes 35 to 37, wherein the CLλ comprises the amino acid sequence according to SEQ ID NO: 107, and the corresponding C H1 It contains the amino acid sequence according to SEQ ID NO: 106.

[0263] Implementation Scheme 39. The binding protein according to any one of Implementation Schemes 1 to 38, wherein the tumor-associated antigen (TAA) is CD20, phosphatidylinositol proteoglycan-3 (GPC3), or containing a leucine-rich repeat sequence 15 (LRRC15).

[0264] Implementation Scheme 40. The binding protein according to any one of Implementation Schemes 1 to 39, wherein the TAA binding domain binds to CD20 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), respectively, comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0265] Implementation Scheme 41. The binding protein according to Implementation Scheme 40, wherein the TAA binding domain comprises 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% of the same V as those in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. H1 Domain and V L A structural domain; or comprising a VH chain according to SEQ ID NO: 7 and a light chain according to SEQ ID NO: 8.

[0266] Implementation Scheme 42. The binding protein according to any one of Implementation Schemes 1 to 35, wherein the TAA binding domain binds to GPC3 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0267] Implementation Scheme 43. The binding protein according to Implementation Scheme 36, wherein the TAA binding domain comprises 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% of the same V as those in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. H1 Domain and V L A structural domain; or comprising a VH chain according to SEQ ID NO: 19 and a light chain according to SEQ ID NO: 20.

[0268] Implementation Scheme 44. The binding protein according to any one of Implementation Schemes 1 to 39, wherein the TAA binding domain binds to LRRC15 and comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), respectively, comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0269] Implementation Scheme 45. The binding protein according to Implementation Scheme 44, wherein the TAA binding domain comprises 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% of the same V as SEQ ID NO: 31 and SEQ ID NO: 32, respectively. H1 Domain and V L A structural domain; or comprising a VH chain according to SEQ ID NO: 31 and a light chain according to SEQ ID NO: 32.

[0270] Implementation Scheme 46. The binding protein according to any one of Implementation Schemes 1 to 45, said binding protein comprising: (a) the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 12; (b) the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24; or (c) the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36.

[0271] Implementation Scheme 47. The binding protein according to Implementation Scheme 46, wherein the first polypeptide chain and the second polypeptide chain comprise the amino acid sequence of SEQ ID NO: 9, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12.

[0272] Implementation Scheme 48. The binding protein according to Implementation Scheme 46, wherein the first polypeptide chain and the second polypeptide chain comprise the amino acid sequence of SEQ ID NO: 21, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24.

[0273] Implementation Scheme 49. The binding protein according to Implementation Scheme 46, wherein the first polypeptide chain and the second polypeptide chain comprise the amino acid sequence of SEQ ID NO: 33, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36.

[0274] Implementation Scheme 50. A pharmaceutical composition comprising a binding protein according to any one of Implementation Schemes 1 to 49, and a pharmaceutically acceptable carrier.

[0275] Implementation Scheme 51. An isolated nucleic acid molecule, said isolated nucleic acid molecule encoding a binding protein according to any one of Implementation Schemes 1 to 49.

[0276] Implementation Scheme 52. A vector comprising isolated nucleic acid molecules according to Implementation Scheme 51.

[0277] Implementation Scheme 53. A method of treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a binding protein according to any one of Implementation Schemes 1 to 49 or a pharmaceutical composition according to Implementation Scheme 50.

[0278] Implementation Scheme 54. A method for treating an inflammatory disease and / or autoimmune condition in a subject of need, the method comprising administering to the subject a therapeutically effective amount of a binding protein according to any one of Implementation Schemes 1 to 49 or a pharmaceutical composition according to Implementation Scheme 50.

[0279] Implementation Scheme 55. The method according to Implementation Scheme 53 or Implementation Scheme 54, wherein the binding protein preferentially activates a subset of the subject's T cells.

[0280] Implementation Scheme 56. The method according to Implementation Scheme 55, wherein the subset of T cells is CD8+ T cells.

[0281] Implementation Scheme 57. The method according to Implementation Scheme 56, wherein the CD8+ T cells are preferentially activated compared to CD4+ T cells.

[0282] Implementation Scheme 58. The method according to any one of Implementation Schemes 53 to 57, wherein the activation of T cells is determined by measuring the percentage of surface CD25+ T cells.

[0283] Implementation Scheme 59. The method according to Implementation Scheme 58, wherein the percentage of surface CD25+ T cells as CD8+ T cells is higher than the percentage of surface CD25+ T cells as CD4+ T cells.

[0284] Implementation Scheme 60. A method for treating an inflammatory disease and / or autoimmune condition in a subject of need, the method comprising administering to the subject a therapeutically effective amount of a binding protein according to any one of Implementation Schemes 1 to 49 or a pharmaceutical composition according to Implementation Scheme 50.

[0285] Implementation Scheme 61. The method according to Implementation Scheme 53, wherein the tumor-associated antigen of the binding protein is CD20 and the cancer is a B-cell malignancy.

[0286] Implementation Scheme 62. The method according to Implementation Scheme 61, wherein the B-cell malignancy is non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, or primary mediastinal large B-cell lymphoma (PMBCL).

[0287] Implementation Scheme 63. The method according to Implementation Scheme 53, wherein the tumor-associated antigen of the binding protein is phosphatidylinositol proteoglycan-3 (GPC3), and the cancer is liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer (sqNSCLC), ovarian cancer, clear cell ovarian cancer, carcinoma, Merkel cell carcinoma, gastric cancer, hepatoblastoma, or nephroblastoma.

[0288] Implementation Scheme 64. The method according to Implementation Scheme 53, wherein the tumor-associated antigen of the binding protein is a leucine-rich repeat sequence 15 (LRRC15), and the cancer is melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, or thyroid cancer.

[0289] Implementation Scheme 65. The inflammatory disease and / or autoimmune condition described in the method according to Implementation Scheme 54 is systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), Sjögren's syndrome, or antineutrophil cytoplasmic autoantibody (ANCA) vasculitis.

[0290] Implementation Scheme 66. A method for treating a subject with systemic lupus erythematosus (SLE) in need, the method comprising administering to the subject a therapeutically effective amount of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50.

[0291] Implementation Scheme 67. A method for treating myositis in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a binding protein according to any one of Implementation Schemes 1 to 49 or a pharmaceutical composition according to Implementation Scheme 50.

[0292] Implementation Scheme 68. A method for treating rheumatoid arthritis (RA) in a subject of need, the method comprising administering to the subject a therapeutically effective amount of a binding protein according to any one of Implementation Schemes 1 to 49 or a pharmaceutical composition according to Implementation Scheme 50.

[0293] Implementation Scheme 69. A method for treating Sjögren's syndrome in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50.

[0294] Implementation Scheme 70. The method according to any one of Implementation Schemes 53 to 69, wherein the binding protein is administered subcutaneously.

[0295] Implementation Scheme 71. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used as a drug.

[0296] Implementation Scheme 72. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used in the treatment of cancer.

[0297] Implementation Scheme 73. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used in the treatment of inflammatory diseases and / or autoimmune diseases.

[0298] Implementation Scheme 74. The binding protein or pharmaceutical composition for use according to Implementation Scheme 72, wherein the tumor-associated antigen is CD20 and the cancer is a B-cell malignancy.

[0299] Implementation Scheme 75. The binding protein or pharmaceutical composition for use according to Implementation Scheme 74, wherein the B-cell malignancy is non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, or primary mediastinal large B-cell lymphoma (PMBCL).

[0300] Implementation Scheme 76. The binding protein or pharmaceutical composition for use according to Implementation Scheme 72, wherein the tumor-associated antigen is phosphatidylinositol proteoglycan-3 (GPC3) and the cancer is hepatocellular carcinoma or hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer (sqNSCLC), ovarian cancer, clear cell ovarian cancer, carcinoma, Merkel cell carcinoma, gastric cancer, hepatoblastoma or nephroblastoma.

[0301] Implementation Scheme 77. The binding protein or pharmaceutical composition for use according to Implementation Scheme 72, wherein the tumor-associated antigen is a leucine-rich repeat sequence 15 (LRRC15), and the cancer is melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, or thyroid cancer.

[0302] Implementation Scheme 78. The binding protein or pharmaceutical composition used according to Implementation Scheme 73, wherein the inflammatory disease and / or autoimmune condition is systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), Sjögren's syndrome, or ANCA vasculitis.

[0303] Implementation Scheme 79. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used in the treatment of systemic lupus erythematosus (SLE).

[0304] Implementation Scheme 80. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used in the treatment of myositis.

[0305] Implementation Scheme 81. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, said binding protein or said pharmaceutical composition for use in the treatment of rheumatoid arthritis (RA).

[0306] Implementation Scheme 82. The binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50, wherein the binding protein or the pharmaceutical composition is used in the treatment of Sjögren's syndrome.

[0307] Implementation Scheme 83. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of cancer.

[0308] Implementation Scheme 84. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of inflammatory and / or autoimmune diseases.

[0309] Implementation Scheme 85. The use according to Implementation Scheme 83, wherein the cancer is a B-cell malignancy, liver cancer, or HCC.

[0310] Implementation Scheme 86. The use according to Implementation Scheme 85, wherein the B-cell malignancy is non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma, or primary mediastinal large B-cell lymphoma (PMBCL).

[0311] Implementation Scheme 87. The use according to Implementation Scheme 83, wherein the cancer is melanoma, sarcoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, osteosarcoma, glioblastoma, lung cancer, non-small cell lung cancer, or thyroid cancer.

[0312] Implementation Scheme 88. The use according to Implementation Scheme 84, wherein the inflammatory and / or autoimmune condition is systemic lupus erythematosus (SLE), myositis, rheumatoid arthritis (RA), Sjögren's syndrome, or ANCA vasculitis.

[0313] Implementation Scheme 89. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of systemic lupus erythematosus (SLE).

[0314] Implementation Scheme 90. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of myositis.

[0315] Implementation Scheme 91. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of rheumatoid arthritis (RA).

[0316] Implementation Scheme 92. Use of the binding protein according to any one of Implementation Schemes 1 to 49 or the pharmaceutical composition according to Implementation Scheme 50 for the manufacture of a medicament for the treatment of Sjögren's syndrome.

[0317] Example The following examples illustrate specific aspects of this disclosure and their various uses. They are shown for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way.

[0318] Example 1: Combining protein structure and sequence This article describes a novel class of binding proteins engineered to improve safety and enhance efficacy. Multiple forms of the binding protein were tested to determine the placement of the TCR-binding domain, the placement of the T cell co-stimulatory domain, the adapter size and cleavability, and the Fc moiety to be used.

[0319] Table 1: LM1486 (CD20), LM1653 (GPC3), LRC150016 (LRRC15), TPP-46956-TPP-46960 Amino acid and nucleic acid sequences of (LRRC15), TPP-42197 (BCMA), and LM1953 (STEAP-2). SEQIDNO: describe sequence 1 LM1486 / LM1486-2CD20VHCDR1 GYTFTSYNMH 2 LM1486 / LM1486-2CD20VHCDR2 AIYPGSGDTSYNQKFKG 3 LM1486 / LM1486-2CD20VHCDR3 VVYYSNSYWYFDV 4 LM1486 / LM1486-2CD20VLCDR1 RASSSVSYMH 5 LM1486 / LM1486-2CD20VLCDR2 APSNLAS 6 LM1486 / LM1486-2CD20VLCDR3 QQWSFNPPT 7 LM1486 / LM1486-2CD20VH EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS 8 LM1486 / LM1486-2CD20VL DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK 9 LM1486 / LM1486-2CD20 Light Chain DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10 LM1486 / LM1486-2CD20VH-CH1 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 11 LM1486-2-juxta (TCR)CD20HCTCR(FC juxta) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 12 LM1486 / LM1486-2-knob (CD8)CD20HC CD8 (Fc knob) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 13 LM1653GPC3VHCDR1 DYEMH 14 LM1653GPC3VHCDR2 ALDPKTGDTAYSQKFKG 15 LM1653GPC3VHCDR3 FYSYTY 16 LM1653GPC3VLCDR1 RSSQSLVHSNRNTYLH 17 LM1653GPC3VLCDR2 KVSNRFS 18 LM1653GPC3VLCDR3 SQNTHVPPT 19 LM1653GPC3VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSS 20 LM1653GPC3VL DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK 21 LM1653GPC3 light chain DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 22 LM1653GPC3VH-CH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 23 LM1653GPC3-Jiu (TCR)HCTCR(FC Jiu) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 24 LM1653 GPC3-knob (CD8) HC CD8 (Fc knob) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 25 LRC150016VHCDR1 SYWIE 26 LRC150016VHCDR2 EILPGSDTTNYNEKFKD 27 LRC150016VHCDR3 DRGNYRAWFGY 28 LRC150016VLCDR1 RASQDISNYLN 29 LRC150016VLCDR2 YTSRLHS 30 LRC150016VLCDR3 QQGEALPWT 31 LRC150016VH EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS 32 LRC150016VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGEALPWTFGGGTKVEIK 33 LRC150016 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGEALPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 34 LRC150016VH-CH1 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 35 LRC150016-ju (TCR)HC(FCju) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 36 LRC150016 - Knob (CD8)HCCD8 (FC Knob) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 Linker TGGS 38 Linker GGGGS 39 <![CDATA[TCRV HHa CDR1]]> INFLG 40 <![CDATA[TCRV HHa CDR2]]> HISIGDQTDYADSAKG 41 <![CDATA[TCRV HHa CDR3]]> FSRIYPYDY 42 <![CDATA[TCRV HHa ]]> EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 43 <![CDATA[TCRhu2.1V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSS 44 <![CDATA[TCRhu2.2V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSS 45 <![CDATA[TCRhu2.3V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSS 46 <![CDATA[TCRhu2.4V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSS 47 <![CDATA[TCRhu2.5V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 48 <![CDATA[TCRhu2.6V HHa ]]> EVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 49 <![CDATA[CD8V HHb CDR1]]> DYAIG 50 <![CDATA[CD8V HHb CDR2]]> IRVSDGSTYYADSVKG 51 <![CDATA[CD8V HHb CDR3]]> GSLYTCVQSIVWPARPYYDMDY 52 <![CDATA[CD8V HHb LM1486(CD20CD8)BCMACD8LM1953(STEAP2CD8)LRC150016(LRRC15)]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 53 IgG1 hinge sequence DKTHTCPPCPAP 54 IgG1 split hinge 1 DK 55 IgG1 split hinge 2 THTCPPCPAP 56 <![CDATA[Fc a (mortar)]]> PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57 <![CDATA[Fc a (Mortar)(RF)]]> PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 58 <![CDATA[Fc b (Pound)]]> PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 59 Framework (TCR / 臼) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 60 Framework (TCR / 臼 / RF) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 61 Scaffold (CD8 / knob) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 62 <![CDATA[TCRV HHa hu2.1,2.3CDR1]]> INFLS 63 <![CDATA[LRC150016-Jaw (TCR)HCTCR(FC Jaw)hu2.1V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKEREKVAHISIGDQTD YADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 64 <![CDATA[LRC150016-Jaw (TCR) HCTCR (FC Jaw) hu2.2V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTD YADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 65 <![CDATA[LRC150016-Joint (TCR)HCTCR(FC Joint)hu2.3V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKGLEKVAHISIGDQTD YADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 66 <![CDATA[LRC150016-臼(TCR)HCTCR(FC臼)hu2.4V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTD YADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 67 <![CDATA[LRC150016-臼(TCR)HCTCR(FC臼)hu2.5V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTD YADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 68 <![CDATA[LRC150016-Jade (TCR) HCTCR (FC Jade) hu2.6V HHa > EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTD YADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 69 <![CDATA[Skeleton (TCR / pocket) TCRhu2.1V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 70 <![CDATA[Scaffold (TCR / pit) TCRhu2.2V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 71 <![CDATA[Scaffold (TCR / pocket) TCRhu2.3V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 72 <![CDATA[Scaffold (TCR / pocket) TCRhu2.4V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPEVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 73 <![CDATA[Scaffold (TCR / pocket) TCRhu2.5V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 74 <![CDATA[Scaffold (TCR / pit) TCRhu2.6V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 75 <![CDATA[Scaffold (TCR / pit / RF) TCRhu2.1V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 76 <![CDATA[Scaffold (TCR / pit / RF) TCRhu2.2V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 77 <![CDATA[Framework (TCR / pocket / RF) TCRhu2.3V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLSWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 78 <![CDATA[Scaffold (TCR / pit / RF) TCRhu2.4V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTLYLQMNSLRAEDTAVYYCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 79 <![CDATA[Scaffold (TCR / pit / RF) TCRhu2.5V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 80 <![CDATA[Scaffold (TCR / pit / RF) TCRhu2.6V HHa > ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKGLEKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRAEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPCPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 81 <![CDATA[CD8V HHb D30P / S75A / Q106N]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVNSIVWPARPYYDMDYWGQGTLVTVSS 82 <![CDATA[CD8V HHb D30P / S75A / L101A]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSAYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 83 <![CDATA[CD8V HHb S75A / D30R]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 84 <![CDATA[CD8V HHb D30P / S75A / S100G]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGGLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 85 <![CDATA[CD8V HHb D30P / S75A / Y102I]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLITCVQSIVWPARPYYDMDYWGQGTLVTVSS 86 <![CDATA[CD8V HHb D30P / S75A / S100P]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGPLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 87 <![CDATA[CD8V HHb S75A / D31S]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 88 <![CDATA[CD8V HHb D30P / S75A / R52T]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCITVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 89 <![CDATA[CD8V HHb D30P / S75A]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFPDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 90 IgG1 IMGT upper hinge region EPKSCDK 91 IMGT lower hinge region THTCPPCP 92 LM1486 CD20 VH-CH1 (IMGT) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV 93 LM1653 GPC3 VH-CH1 (IMGT) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV 94 LRC150016 VH-CH1 (IMGT) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV 95 <![CDATA[WT C H1 ]]> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 96 <![CDATA[WT C H1 (IMGT)]]> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV 97 <![CDATA[V12C H1 (F126C / C220V)]]> ASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSV 98 WT CLλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 99 V12LCλ constant (CLλ) GQPKAAPSVTLFPPCSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS 100 WT LCκ constant (Cκ) region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 101 IgG1 CH2 wild-type ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 102 IgG1 CH2 Fc-inactivated PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 103 Wild-type IgG1 CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 104 <![CDATA[C with A141S H1 > ASTKGPSVFPLAPSSKSTSGGTASLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 105 CLλ with T117R GQPKAAPSVRLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 106 <![CDATA[V12C with A141S H1 > ASTKGPSVCPLAPSSKSTSGGTASLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSV 107 V12 CLλ with T117R GQPKAAPSVRLFPPCSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS 108 IgG1 CH2 WT (IMGT) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 109 IgG1 CH2 Fc-inactive (IMGT) APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 110 LM1486 CD20 VH-CH-TCR-CH2CH3 (knob / RF) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGSGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 111 TPP-42197BCMAVHCDR1 SYSMN 112 TPP-42197BCMAVHCDR2 SISGQSNYIYYADSVKG 113 TPP-42197BCMAVHCDR3 GGNYFVEYFQY 114 TPP-42197BCMAVLCDR1 RASQYISSNNLA 115 TPP-42197BCMAVLCDR2 GASNRAT 116 TPP-42197BCMAVLCDR3 QQYADSPIT 117 TPP-42197BCMAVH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQYWGQGTLVTVSS 118 TPP-42197BCMAVL EIVLTQSPGTLSLSPGERATLSCRASQYISSNNLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTKLEIK 119 TPP-42197 BCMA light chain EIVLTQSPGTLSLSPGERATLSCRASQYISSNNLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 120 TPP-42197 BCMA VH-CH1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 121 TPP-42197 mouse (TCR) BCMA heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 122 TPP-42197-杵(CD8) BCMA HC EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 123 LM1953STEAP2 HCCDR1 RNSAVWN 124 LM1953STEAP2 HCCDR2 RTYYRSKWYNDYAVSVKS 125 LM1953STEAP2 HCCDR3 GLRQNQFYYYMDV 126 LM1953STEAP2 LCCDR1 RASQSVASNLA 127 LM1953STEAP2 LCCDR2 GASTRAT 128 LM1953STEAP2 LCCDR3 QQYNNWPFT 129 LM1953STEAP2 VH QVQLQQSGPGLVKPSQTLSLTCAISGDSVSRNSAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGLRQNQFYYYMDVWGKGTTVTVSS 130 LM1953STEAP2 VL EIVMTQSPATLSVSPGERATLSCRASQSVASNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPFTFGPGTKVDIK 131 LM1953 STEAP2 LC EIVMTQSPATLSVSPGERATLSCRASQSVASNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 132 LM1953 STEAP2 VH-CH1 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSRNSAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGLRQNQFYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 133 LM1953 STEAP2 knob (CD8) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSRNSAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGLRQNQFYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 134 LM1953 STEAP2 epitope (TCR) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSRNSAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGLRQNQFYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 135 TPP-46956TPP-46957TPP-46958TPP-46959TPP-46960TPP-47826TPP-49058LRRC15 VHCDR1 SYWIE 136 TPP-46956LRRC15 VHCDR2 EILPGSDTTNYAQNFQD 137 TPP-46956TPP-46957TPP-46958TPP-46959TPP-46960TPP-47826TPP-49058LRRC15 VHCDR3 DRGNYRAWFGY 138 TPP-46956TPP-46957TPP-46958TPP-46959TPP-46960TPP-47826TPP-49058LRRC15 VLCDR1 RASQDISNYLN 139 TPP-46956TPP-46957TPP-46958TPP-47826LRRC15 VLCDR2 YTSRLHS 140 TPP-46956TPP-46957TPP-46958TPP-46959TPP-46960TPP-47826TPP-49058LRRC15 VLCDR3 QQGNALPWT 141 TPP-46956LRRC15 VH EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQNFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS 142 TPP-46956TPP-46957TPP-46958TPP-47826LRRC15 VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIK 143 TPP-46956TPP-46957TPP-46958TPP-47826LRRC15 light chain DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 144 TPP-46956LRRC15 VH-CH1 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQNFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 145 TPP-46956LRRC15臼(TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQNFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 146 TPP-46956LRRC15 Knob (CD8) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQNFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 147 TPP-46957LRRC15 VHCDR2 EILPGSDTTNYAQQFQD 148 TPP-46957LRRC15 VH EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQQFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS 149 TPP-46957LRRC15 VH-CH1 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQQFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 150 TPP-46957 LRRC15 knob (TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQQFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 151 TPP-46957 LRRC15 hole (CD8) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQQFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 152 TPP-46958TPP-47826LRRC15 VHCDR2 EILPGSDATNYAQKFQD 153 TPP-46958TPP-47826LRRC15 VH EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDATNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS 154 TPP-46958TPP-47826LRRC15 VH-CH1 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDATNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 155 TPP-46958 LRRC15 knob (TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDATNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 156 TPP-46958 TPP-47826 LRRC15 hole (CD8) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDATNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 157 TPP-46959TPP-46960TPP-49058LRRC15 VHCDR2 EILPGSDTTNYAQKFQD 158 TPP-46959TPP-49058LRRC15 VLCDR2 YTSRLES 159 TPP-46959TPP-46960TPP-49058LRRC15 VH EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS 160 TPP-46959TPP-49058LRRC15 VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIK 161 TPP-46959TPP-49058LRRC15 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 TPP-46959TPP-46960TPP-49058LRRC15 VH-CH1 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 163 TPP-46959TPP-46960LRRC15臼 (TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 164 TPP-46959TPP-46960TPP-49058LRRC15 knob (CD8) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 165 TPP-46960LRRC15 VLCDR2 YTSRLNS 166 TPP-46960LRRC15 VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLNSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIK 167 TPP-46960LRRC15 light chain DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKFLIYYTSRLNSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNALPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 168 <![CDATA[CD8V HHb D30T / N74S]]> EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAAGSLYTCVQSIVWPARPYYDMDYWGQGTLVTVSS 169 <![CDATA[CD8V HHb D30T / N74SCDR1IMGT]]> GFTFTDYA 170 <![CDATA[CD8V HHb D30T / N74SCDR2IMGT]]> IRVSDGST 171 <![CDATA[CD8V HHb D30T / N74SCDR3IMGT]]> AAGSLYTCVQSIVWPARPYYDMDY 172 <![CDATA[TCRV HHa Substitutes DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSS 173 <![CDATA[TCRV HHa CDR1 alternatives]]> INFYG 174 <![CDATA[TCRV HHa CDR2 alternatives]]> HISIGDQTDYADSAKG 175 <![CDATA[TCRV HHa CDR3 alternatives]]> LSRIWPYDY 176 TPP-47826 LRRC15 mu (TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDATNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSDVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 177 TPP-49058 LRRC15 mu (TCR) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYAQKFQDRVTFTSDTSISTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTGGSDVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 178 Reference THC (CD3) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYCARHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 179 Compare TLC(CD3) QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 180 Compare THC(BCMA) QLOLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 181 Comparator TLC (BCMA) SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 182 LM1486 / LM1486-2CD20VHCDR1 Kabat SYNMH 183 <![CDATA[CD8V HHb CDR2 (alternative) CIRVSDGSTYYADSVKG 184 LM1653 GPC3 Heavy Chain Knob-into-hole (TCR) CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTTACCGACTACGAGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCGCCCTGGATCCTAAGACCGGCGATACCGCTTACTCCCAGAAATTCAAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGACCTCTGAGGACACCGCCGTGTACTACTGCACCCGGTTCTACTCCTACACCTACTGGGGCCAGGGCACCCTGGTTACAGTGTCCTCTGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCATCCTCCAAGTCCACCTCTGGTGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAATACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGACAAGACAGGCGGATCTGAGGTCCAGCTGGTCGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCTAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCAGAGGATACCGCTGTGTATTTCTGCCGGGCCTTCAGCCGGATCTACCCTTACGATTATTGGGGACAGGGAACACTCGTGACAGTGTCTAGCGGAGGCGGAGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCCTCTGATATCGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 185 LM1653 GPC3 Heavy Chain Protrusion (CD8) CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTTACCGACTACGAGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCGCCCTGGATCCTAAGACCGGCGATACCGCTTACTCCCAGAAATTCAAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGACCTCTGAGGACACCGCCGTGTACTACTGCACCCGGTTCTACTCCTACACCTACTGGGGCCAGGGCACCCTGGTTACAGTGTCCTCTGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCATCCTCCAAGTCCACCTCTGGTGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTCGACAAGAGAGTGGAACCCAAGTCCTGCGACAAGACAGGCGGATCTGAGGTGCAGCTGCTCGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCTGCCTCCGGCTTCACCTTCGACGATTACGCCATCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGGGCGTCAGCTGCATCAGAGTGTCTGACGGCTCTACCTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGATACCGCTGTGTATTATTGCGCCGCTGGCTCCCTGTATACCTGCGTGCAGTCTATCGTGTGGCCCGCCAGACCTTACTACGACATGGACTATTGGGGACAGGGAACACTCGTGACAGTGTCTAGCGGAGGCGGCGGATCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 186 LM1653 GPC3 Light Chain GACGTCGTGATGACCCAGTCTCCTCTGTCTCTGCCTGTGACACCTGGCGAGCCTGCCTCCATCTCTTGCAGATCTTCTCAGTCCCTGGTGCACTCCAACCGGAACACCTACCTGCACTGGTATCTGCAGAAGCCCGGACAGTCTCCCCAGCTGCTGATCTACAAGGTGTCCAACAGATTCTCTGGCGTGCCCGACAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCTCTCAGAATACCCACGTGCCACCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCCACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 187 LM1486-2CD20 heavy chain knob (TCR) GAGGTGCAGCTGGTTGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCTGCCTCTGGCTACACCTTCACCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGCAAAGGATTGGAATGGGTCGGAGCTATCTACCCTGGCTCCGGCGATACCTCCTACAACCAGAAGTTCAAGGGCAGATTCACCATCTCCGTGGACAAGTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGTGGTGTACTACTCCAACTCCTACTGGTACTTCGACGTGTGGGGCCAGGGAACACTGGTCACAGTGTCCTCTGCTTCCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGTGGAACCGCTGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCTGAGCCTGTGACCGTGTCCTGGAATTCTGGTGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAAGTCCAGTTGGTGGAAAGCGGAGGTGGACTTGTGCAGCCAGGTGGAAGCCTGAGACTGAGTTGTGTGGCTTCTGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGGCAGGCTCCCGGCAAAGAAAGGGAAAAAGTGGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAAGGCCGGTTTACCATCTCTCGGGACGAGAGCAAGAACATGGTGTATCTCCAGATGAACAGTCTGAAGCCCGAGGATACAGCTGTGTACTTCTGCCGGGCCTTCAGCCGGATCTACCCTTACGATTATTGGGGACAGGGCACCCTGGTTACCGTTTCTAGTGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCACCTGTGGCCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAACACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGCGTGGTGTCTGTGCTGACCGTTCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAAGTCTGTACACTGCCTCCTAGCCGGGAAGAGATGACCAAGAATCAGGTGTCCCTGTCCTGCGCCGTGAAGGGCTTCTACCCTTCTGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACCGTGGATAAGAGCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAATCCCTGTCTCTGAGCCCTGGCAAG 188 LM1486 / LM1486-2CD20 heavy chain peg (CD8) GAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCTGCCTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGCAAAGGATTGGAATGGGTCGGAGCTATCTACCCCGGCTCTGGCGACACCTCCTACAACCAGAAGTTCAAGGGCAGATTCACCATCTCCGTGGACAAGTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAGTGGTGTACTACAGCAACTCCTACTGGTACTTCGACGTGTGGGGCCAGGGCACACTGGTCACAGTTTCTTCCGCCTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCATCCTCCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGAAGCGAAGTGCAGCTGCTTGAAAGCGGAGGTGGACTTGTGCAGCCAGGCGGAAGCCTGAGATTGTCCTGTGCCGCTTCTGGCTTTACCTTCGACGACTACGCCATCGGCTGGTTCAGACAGGCTCCCGGAAAAGAGAGAGAGGGCGTCAGCTGCATCAGAGTGTCTGACGGCTCTACCTACTACGCCGACTCCGTGAAAGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAATACTCTGTATCTCCAAATGAACAGCCTGCGCGCTGAGGATACCGCTGTGTATTATTGCGCCGCTGGCTCCCTGTATACCTGCGTGCAGTCTATCGTGTGGCCCGCCAGACCTTACTACGACATGGATTACTGGGGACAGGGAACCCTGGTTACCGTGTCTAGCGGCGGAGGCGGATCTACCCATACCTGTCCTCCATGTCCTGCTCCACCTGTGGCCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAACACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTTCTGCATCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCACCTTGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGATAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAG 189 LM1486 / LM1486-2CD20 light chain GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACAATCACCTGTAGAGCCTCCAGCTCCGTGTCCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCCTCTGATCTACGCTCCTTCTAATCTGGCCTCCGGCGTGCCCTCTAGATTTTCTGGCTCTGGATCTGGCACCGACTTCACCCTGACCATCAGTTCTCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCTTTCAACCCTCCTACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCCACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGT 190 LM1953 STEAP2 heavy chain knob-in-hole (TCR) CAAGTGCAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCCAGAAACAGTGCTGTTTGGAATTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGGGGGTTACGACAGAACCAGTTCTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCCGCTAGTACCAAGGGACCCAGCGTGTTCCCTCTGGCACCTTCCAGCAAGTCTACCTCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCAGCGGAGTGCATACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGGGTGGAACCCAAGTCCTGCGACAAGACCGGCGGATCTGAAGTGCAGCTGGTCGAGTCTGGCGGAGGATTGGTTCAACCTGGCGGCTCCCTGAGACTGTCTTGTGTGGCTAGCGGAGATGTGCATAAGATCAATTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTGTCATCTGGAGGCGGCGGATCTACTCACACGTGCCCACCGTGCCCAGCACCTCCGGTGGCCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCGCGAACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAAACACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTGCACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAGCTGCGCGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTTAGCAAGCTCACCGTGGACAAGAGCCGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 191 LM1953 STEAP2 heavy chain protuberance (CD8) CAAGTGCAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCCAGAAACAGTGCTGTTTGGAATTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGGGGGTTACGACAGAACCAGTTCTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCCGCTAGCACCAAGGGACCCAGCGTGTTCCCTCTGGCACCTTCCAGCAAGTCTACCTCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCAGCGGAGTGCATACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGGGTGGAACCCAAGTCCTGCGACAAGACCGGCGGATCTGAGGTCCAATTGTTGGAATCTGGAGGGGGGCTCGTGCAGCCGGGGGGAAGTTTGCGCCTTTCATGCGCTGCATCAGGGTTTACTTTCGACGACTACGCTATTGGCTGGTTCAGGCAAGCGCCGGGCAAGGAACGCGAAGGCGTGTCCTGCATAAGAGTTTCAGATGGCAGTACCTATTACGCCGATAGCGTCAAGGGACGATTCACGATCAGCCGGGATAATAGCAAAAATACACTTTATTTGCAAATGAACTCTCTTAGGGCAGAAGATACTGCGGTCTATTATTGTGCGGCCGGGTCCCTTTATACCTGCGTGCAGTCTATCGTATGGCCTGCTAGGCCCTATTACGATATGGATTACTGGGGACAGGGTACACTCGTGACGGTATCCAGTGGAGGCGGCGGATCTACTCACACGTGCCCACCGTGCCCAGCACCTCCGGTGGCCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGTACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAAACACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATGCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCCGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCTTAAGCCTGTCTCCGGGTAAA 192 LM1953 STEAP2 light chain GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCCGGGGAAAGAGCCACCCTCTCCTGCCGAGCCAGTCAGAGTGTTGCCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGAGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAACAGTATAATAACTGGCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 193 TPP-42197-BCMA Heavy Chain Knob (CD8) GAGGTGCAGCTGGTGGAAAGTGGAGGTGGCCTGGTTAAGCCTGGCGGGAGCCTGCGCCTGAGCTGCGCTGCCAGCGGCTTCACTTTCTCATCTTACAGCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTTGAGTGGGTTAGTAGCATCAGCGGCCAGTCAAACTACATTTATTACGCCGATTCCGTCAAGGGCAGGTTTACAATCTCCCGTGATAATGCGAAGAATAGCCTCTATCTCCAGATGAATTCCCTGCGCGCCGAAGATACAGCCGTGTATTACTGCGCGAGGGGCGGGAATTACTTTGTTGAATACTTCCAGTACTGGGGCCAGGGTACCCTCGTCACCGTTTCCAGTGCAAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAACCAAAAAGCTGCGACAAGACCGGAGGATCTGAGGTCCAGCTGCTAGAGTCTGGCGGAGGATTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCTGGCTTCACCTTCGACGATTACGCCATCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGGGCGTCAGCTGCATCAGAGTGTCTGACGGCTCCACCTACTACGCCGATTCTGTGAAGGGCAGATTCACCATCTCCCGCGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGTGCTGCCGGCTCTCTGTACACCTGTGTGCAGTCTATCGTGTGGCCCGCCAGACCTTACTACGACATGGACTATTGGGGCCAGGGCACCCTAGTGACCGTCTCAAGCGGTGGCGGAGGATCTACCCACACCTGTCCTCCATGCCCTGCTCCACCCGTGGCCGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCACCTTGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGAAAA 194 TPP-42197-BCMA Heavy Chain Hole (TCR) GAGGTGCAGCTGGTGGAAAGTGGAGGTGGCCTGGTTAAGCCTGGCGGGAGCCTGCGCCTGAGCTGCGCTGCCAGCGGCTTCACTTTCTCATCTTACAGCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTTGAGTGGGTTAGTAGCATCAGCGGCCAGTCAAACTACATTTATTACGCCGATTCCGTCAAGGGCAGGTTTACAATCTCCCGTGATAATGCGAAGAATAGCCTCTATCTCCAGATGAATTCCCTGCGCGCCGAAGATACAGCCGTGTATTACTGCGCGAGGGGCGGGAATTACTTTGTTGAATACTTCCAGTACTGGGGCCAGGGTACCCTCGTCACCGTTTCCAGTGCAAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAACCAAAAAGCTGCGACAAGACCGGAGGATCTGAAGTGCAGCTGGTCGAGTCTGGCGGAGGATTGGTTCAACCTGGCGGCTCCCTGAGACTGTCTTGTGTGGCTAGCGGAGATGTGCATAAGATCAATTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTGTCATCTGGTGGCGGAGGATCTACCCACACCTGTCCTCCATGCCCTGCTCCACCCGTGGCCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAAGTCTGTACACTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCTTGCGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCGGTTCACCCAGAAATCCCTGTCTCTGTCCCCTGGCAAG 195 TPP-42197-BCMA Light Chain GAGATTGTCCTGACACAGTCCCCTGGCACCCTTAGCCTGTCCCCCGGTGAAAGAGCGACCCTGTCCTGCCGTGCCTCACAATACATCTCTTCCAACAATCTGGCGTGGTACCAACAGAAGCCCGGGCAAGCTCCCCGCCTCCTGATTTACGGGGCATCCAATAGAGCCACCGGGATCCCTGATAGATTCTCCGGCAGCGGTAGCGGTACCGATTTCACCCTGACCATCTCTCGCCTGGAGCCTGAAGACTTCGCGGTGTACTATTGTCAACAGTATGCTGATTCCCCAATCACATTCGGCCAGGGTACTAAGCTTGAAATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 196 LRC150016 LRRC15 Heavy Chain Knob (CD8) GAAGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCCGGCTACAAGTTCTCCAGCTACTGGATCGAGTGGGTCAAGCAGGCTCCTGGACAGGGACTCGAGTGGATCGGAGAGATCCTGCCTGGCTCTGACACCACCAACTACAACGAGAAGTTCAAGGACCGGGCCACCTTCACCTCCGACACCTCTATCAACACCGCCTACATGGAACTGTCCCGGCTGAGATCTGACGACACCGCCGTGTACTACTGCGCCAGAGACAGAGGCAACTACAGAGCTTGGTTTGGCTACTGGGGCCAGGGCACACTGGTCACAGTTTCTTCTGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGTTGGAATCTGGCGGTGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCTTCTGGCTTCACCTTCGACGACTACGCCATCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGGGCGTCAGCTGCATCAGAGTGTCTGACGGCTCTACCTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTGCTGGCTCCCTGTACACCTGTGTGCAGTCTATCGTGTGGCCCGCCAGACCTTACTACGACATGGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 197 LRC150016 LRRC15 Heavy Chain Hole (TCR) GAAGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCCGGCTACAAGTTCTCCAGCTACTGGATCGAGTGGGTCAAGCAGGCTCCTGGACAGGGACTCGAGTGGATCGGAGAGATCCTGCCTGGCTCTGACACCACCAACTACAACGAGAAGTTCAAGGACCGGGCCACCTTCACCTCCGACACCTCTATCAACACCGCCTACATGGAACTGTCCCGGCTGAGATCTGACGACACCGCCGTGTACTACTGCGCCAGAGACAGAGGCAACTACAGAGCTTGGTTTGGCTACTGGGGCCAGGGCACACTGGTCACAGTTTCTTCTGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 198 LRC150016 LRRC15 Light Chain GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAACCTGGCGGCGCTGTGAAGTTCCTGATCTACTACACCTCTCGGCTGCACTCCGGCGTGCCCTCTAGATTTTCTGGCTCTGGATCCGGCACCGACTATACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCTACCTACTTCTGCCAGCAAGGCGAGGCTCTGCCTTGGACATTTGGCGGCGGAACAAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 199 TPP-46956 LRRC15 heavy chain knob (CD8) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 200 TPP-46956 LRRC15 heavy chain hole (TCR) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 201 TPP-46956 LRRC15 light chain GACATCCAGATGACCCAGAGTCCCTCTAGTCTCTCAGCTTCCGTGGGGGACCGCGTCACTATAACTTGCCGTGCCTCACAAGACATATCAAACTACCTCAACTGGTATCAACAAAAGCCTGGAAAAGCTCCTAAGTTTTTGATTTATTACACTTCCAGGCTGGAGTCCGGTGTCCCATCCAGGTTCAGCGGCAGTGGAAGCGGAACCGACTATACACTGACTATTTCTAGCTTGCAACCCGAGGACTTTGCCACCTATTATTGCCAGCAAGGTAACGCCCTGCCCTGGACCTTCGGTGGCGGAACCAAGGTGGAAATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 202 TPP-46957 LRRC15 heavy chain knob (CD8) GAGGTTCAGTTGGTACAATCTGGAGCCGAGGTGAAAAAACCAGGTGCAAGCGTCAAGGTTTCCTGCAAAGCTAGTGGGTACAAATTCAGTTCATACTGGATAGAGTGGGTTAAGCAAGCTCCTGGACAAGGTTTGGAGTGGATTGGTGAAATCTTGCCTGGCAGCGATACTACAAACTACGCTCAGCAGTTTCAGGACCGCGTAACATTCACCTCTGATACATCTATATCAACTGCATATATGGAACTTAGCAGGCTCAGGTCCGACGACACTGCCGTCTATTACTGCGCTCGGGATAGGGGGAATTATCGTGCTTGGTTTGGTTATTGGGGTCAAGGGACACTGGTTACAGTTTCTTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 203 TPP-46957 LRRC15 heavy chain hole (TCR) GAGGTTCAGTTGGTACAATCTGGAGCCGAGGTGAAAAAACCAGGTGCAAGCGTCAAGGTTTCCTGCAAAGCTAGTGGGTACAAATTCAGTTCATACTGGATAGAGTGGGTTAAGCAAGCTCCTGGACAAGGTTTGGAGTGGATTGGTGAAATCTTGCCTGGCAGCGATACTACAAACTACGCTCAGCAGTTTCAGGACCGCGTAACATTCACCTCTGATACATCTATATCAACTGCATATATGGAACTTAGCAGGCTCAGGTCCGACGACACTGCCGTCTATTACTGCGCTCGGGATAGGGGGAATTATCGTGCTTGGTTTGGTTATTGGGGTCAAGGGACACTGGTTACAGTTTCTTCAGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 204 TPP-46957 LRRC15 light chain GATATTCAGATGACACAATCCCCTAGCAGTCTCAGTGCCTCAGTAGGGGACCGCGTCACAATAACATGTAGAGCCTCCCAGGATATTTCTAACTACTTGAACTGGTACCAGCAAAAGCCCGGTAAAGCCCCTAAATTCCTTATTTACTACACAAGCCGATTGCACTCTGGTGTACCATCCAGGTTTAGTGGAAGCGGCTCTGGTACAGATTACACACTGACCATTTCATCCCTGCAACCAGAGGATTTCGCCACATATTACTGCCAACAGGGAAACGCCCTCCCTTGGACCTTTGGTGGGGGAACTAAAGTTGAGATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 205 TPP-46958 LRRC15 Heavy Chain Knob (CD8) GAGGTGCAGCTTGTTCAAAGCGGTGCAGAGGTTAAGAAGCCTGGAGCTAGCGTAAAAGTGTCCTGCAAAGCTAGTGGGTATAAGTTCTCTAGCTACTGGATCGAGTGGGTGAAACAAGCCCCAGGGCAGGGGTTGGAGTGGATTGGAGAAATACTCCCCGGATCAGACGCCACTAACTATGCACAGAAGTTTCAAGACAGGGTTACCTTCACATCAGACACATCCATATCAACCGCATATATGGAGTTGTCCAGGCTGCGGAGCGACGACACCGCTGTCTATTATTGTGCCAGGGACCGGGGGAACTACAGGGCTTGGTTTGGGTACTGGGGACAGGGTACACTCGTCACAGTGTCCTCCGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 206 TPP-46958 LRRC15 Heavy Chain Hole (TCR) GAGGTGCAGCTTGTTCAAAGCGGTGCAGAGGTTAAGAAGCCTGGAGCTAGCGTAAAAGTGTCCTGCAAAGCTAGTGGGTATAAGTTCTCTAGCTACTGGATCGAGTGGGTGAAACAAGCCCCAGGGCAGGGGTTGGAGTGGATTGGAGAAATACTCCCCGGATCAGACGCCACTAACTATGCACAGAAGTTTCAAGACAGGGTTACCTTCACATCAGACACATCCATATCAACCGCATATATGGAGTTGTCCAGGCTGCGGAGCGACGACACCGCTGTCTATTATTGTGCCAGGGACCGGGGGAACTACAGGGCTTGGTTTGGGTACTGGGGACAGGGTACACTCGTCACAGTGTCCTCCGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 207 TPP-46958 LRRC15 Light Chain GATATTCAGATGACACAATCCCCTAGCAGTCTCAGTGCCTCAGTAGGGGACCGCGTCACAATAACATGTAGAGCCTCCCAGGATATTTCTAACTACTTGAACTGGTACCAGCAAAAGCCCGGTAAAGCCCCTAAATTCCTTATTTACTACACAAGCCGATTGCACTCTGGTGTACCATCCAGGTTTAGTGGAAGCGGCTCTGGTACAGATTACACACTGACCATTTCATCCCTGCAACCAGAGGATTTCGCCACATATTACTGCCAACAGGGAAACGCCCTCCCTTGGACCTTTGGTGGGGGAACTAAAGTTGAGATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 208 TPP-46959 LRRC15 heavy chain knob (CD8) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 209 TPP-46959 LRRC15 heavy chain hole (TCR) AGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 210 TPP-46959 LRRC15 light chain GACATCCAGATGACCCAGAGTCCCTCTAGTCTCTCAGCTTCCGTGGGGGACCGCGTCACTATAACTTGCCGTGCCTCACAAGACATATCAAACTACCTCAACTGGTATCAACAAAAGCCTGGAAAAGCTCCTAAGTTTTTGATTTATTACACTTCCAGGCTGGAGTCCGGTGTCCCATCCAGGTTCAGCGGCAGTGGAAGCGGAACCGACTATACACTGACTATTTCTAGCTTGCAACCCGAGGACTTTGCCACCTATTATTGCCAGCAAGGTAACGCCCTGCCCTGGACCTTCGGTGGCGGAACCAAGGTGGAAATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 211 TPP-46960 LRRC15 Heavy Chain Knob (CD8) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 212 TPP-46960 LRRC15 Heavy Chain Hole (TCR) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGTACTAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGTGGCTAGCGGCGACGTGCACAAGATCAACTTTCTCGGCTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGAAGGTCGCCCACATCTCCATCGGCGACCAGACCGATTACGCCGACTCTGCCAAGGGCAGATTCACCATCTCTCGGGACGAGTCCAAGAACATGGTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCCGGGCCTTCTCTCGGATCTACCCCTACGATTATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGCGGAGGCGGCTCTACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAAGTCTGTACCCTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACAGGTTCACCCAGAAGTCCCTGTCTCTGAGCCCTGGCAAG 213 TPP-46960 LRRC15 Light Chain GACATACAGATGACTCAGTCTCCATCTTCCCTTTCCGCTTCTGTGGGTGATCGAGTAACAATTACATGCCGGGCATCACAGGATATTTCTAACTATCTTAATTGGTATCAACAAAAACCAGGTAAGGCTCCAAAGTTTCTGATCTATTATACCTCTCGGCTGAACAGTGGTGTCCCCAGTAGGTTCAGCGGTAGTGGCAGCGGAACTGACTACACCCTGACCATTTCTTCTCTGCAACCCGAAGATTTTGCAACCTACTATTGTCAACAAGGCAACGCTCTTCCTTGGACCTTTGGCGGAGGCACAAAGGTCGAAATAAAACGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 214 TPP-47826 LRRC15 heavy chain knob (CD8) GAGGTGCAGCTTGTTCAAAGCGGTGCAGAGGTTAAGAAGCCTGGAGCTAGCGTAAAAGTGTCCTGCAAAGCTAGTGGGTATAAGTTCTCTAGCTACTGGATCGAGTGGGTGAAACAAGCCCCAGGGCAGGGGTTGGAGTGGATTGGAGAAATACTCCCCGGATCAGACGCCACTAACTATGCACAGAAGTTTCAAGACAGGGTTACCTTCACATCAGACACATCCATATCAACCGCATATATGGAGTTGTCCAGGCTGCGGAGCGACGACACCGCTGTCTATTATTGTGCCAGGGACCGGGGGAACTACAGGGCTTGGTTTGGGTACTGGGGACAGGGTACACTCGTCACAGTGTCCTCCGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 215 TPP-47826 LRRC15 heavy chain hole (TCR) GAAGTGCAGTTGGTGCAGTCTGGGGCCGAAGTTAAGAAACCAGGGGCCTCTGTTAAAGTAAGTTGCAAGGCTTCCGGTTACAAGTTTTCAAGCTACTGGATCGAGTGGGTCAAGCAGGCCCCAGGCCAAGGGTTGGAATGGATAGGAGAGATCCTTCCAGGGAGCGATGCCACAAACTACGCCCAAAAATTCCAGGACAGGGTTACTTTTACTAGTGACACTTCCATATCAACAGCCTATATGGAGCTGTCCCGATTGCGCTCCGATGATACTGCCGTGTACTATTGCGCCAGGGATAGAGGCAATTACCGCGCATGGTTTGGATACTGGGGGCAGGGAACACTTGTGACAGTTAGCTCCGCAAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAACCAAAAAGCTGTGATAAAACCGGAGGTTCAGACGTGCAGTTGGTGGAATCTGGAGGCGGTGTTGTACAGCCCGGCGGCTCTTTGAGACTCTCATGTGTCGCCAGCGGCTACGTACACAAGATCAATTTTTATGGCTGGTATAGGCAAGCTCCCGGTAAAGAGCGCGAGAAAGTCGCTCATATTAGCATCGGAGATCAGACTGACTACGCCGACTCCGCTAAAGGGCGCTTCACCATATCTAGGGATGAATCAAAGAACACTGTGTATCTCCAGATGAACTCTCTCCGCCCAGAGGATACTGCCGCTTACTATTGTCGTGCTCTTTCCCGTATATGGCCTTATGATTACTGGGGACAGGGGACATTGGTTACTGTGAGTTCTGGTGGCGGAGGATCTACCCACACCTGTCCTCCATGCCCTGCTCCACCCGTGGCCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAAGTCTGTACACTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCTTGCGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCGGTTCACCCAGAAATCCCTGTCTCTGTCCCCTGGCAAG 216 TPP-47826 LRRC15 light chain GATATTCAGATGACACAATCCCCTAGCAGTCTCAGTGCCTCAGTAGGGGACCGCGTCACAATAACATGTAGAGCCTCCCAGGATATTTCTAACTACTTGAACTGGTACCAGCAAAAGCCCGGTAAAGCCCCTAAATTCCTTATTTACTACACAAGCCGATTGCACTCTGGTGTACCATCCAGGTTTAGTGGAAGCGGCTCTGGTACAGATTACACACTGACCATTTCATCCCTGCAACCAGAGGATTTCGCCACATATTACTGCCAACAGGGAAACGCCCTCCCTTGGACCTTTGGTGGGGGAACTAAAGTTGAGATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 217 TPP-49058 LRRC15 heavy chain knob (CD8) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCCAAGTCCTGCGATAAGACCGGCGGATCTGAGGTACAGCTGCTGGAATCAGGAGGCGGCTTGGTTCAGCCAGGAGGCTCACTTCGGCTCTCCTGCGCAGCAAGTGGGTTTACATTTACTGATTATGCTATAGGCTGGTTTCGGCAAGCACCTGGTAAGGAACGCGAAGGTGTGTCATGTATTCGCGTAAGCGACGGATCAACATACTATGCAGACAGTGTCAAAGGTAGATTTACTATTAGTCGTGACAGCTCAAAGAATACTCTGTACTTGCAAATGAATTCCCTTCGTGCCGAAGATACTGCCGTTTACTATTGCGCCGCAGGATCACTCTACACCTGCGTGCAGAGCATTGTTTGGCCTGCCAGGCCTTATTATGATATGGACTATTGGGGCCAAGGCACTTTGGTCACTGTTAGCTCTGGAGGCGGAGGCAGCACCCATACCTGTCCTCCATGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGACAGCCCAGGGAACCCCAGGTTTACACCCTGCCTCCATGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTTAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGAGCCCCGGCAAA 218 TPP-49058 LRRC15 heavy chain hole (TCR) GAGGTGCAGTTGGTCCAATCAGGAGCAGAAGTCAAAAAACCCGGTGCTTCCGTTAAAGTATCTTGCAAAGCTAGCGGGTACAAGTTCTCTTCCTATTGGATCGAGTGGGTTAAGCAGGCACCTGGACAGGGTCTTGAATGGATAGGCGAAATCCTTCCCGGTAGCGACACCACAAACTATGCTCAAAAGTTCCAAGATAGAGTTACTTTCACTTCAGATACTTCCATATCTACTGCATATATGGAACTGTCCCGTTTGAGGAGTGATGACACAGCAGTGTACTACTGTGCCCGTGACCGTGGAAACTACCGTGCATGGTTCGGCTATTGGGGTCAGGGTACACTCGTAACAGTGAGCTCAGCAAGCACCAAGGGACCCTCTGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAACCAAAAAGCTGTGACAAGACTGGTGGCTCAGACGTACAATTGGTTGAAAGTGGTGGGGGCGTAGTCCAGCCCGGAGGCAGCCTCCGACTTTCCTGCGTCGCATCTGGATACGTCCATAAAATCAACTTTTACGGCTGGTACAGACAAGCACCAGGGAAAGAGCGGGAAAAAGTTGCCCACATATCCATTGGCGACCAGACAGATTATGCCGACAGCGCCAAGGGTCGATTTACTATAAGTCGGGACGAATCAAAGAATACTGTGTATCTCCAGATGAACAGTCTTCGACCTGAAGACACCGCCGCTTATTATTGTAGGGCTTTGTCCAGGATCTGGCCATACGATTACTGGGGGCAAGGGACACTCGTAACCGTTAGTTCTGGTGGCGGAGGATCTACCCACACCTGTCCTCCATGCCCTGCTCCACCCGTGGCCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGAAGCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGGCTAAGGGCCAGCCTCGCGAGCCTCAAGTCTGTACACTGCCTCCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGTCTTGCGCCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCGGTTCACCCAGAAATCCCTGTCTCTGTCCCCTGGCAAG 219 TPP-49058 LRRC15 light chain GACATCCAGATGACCCAGAGTCCCTCTAGTCTCTCAGCTTCCGTGGGGGACCGCGTCACTATAACTTGCCGTGCCTCACAAGACATATCAAACTACCTCAACTGGTATCAACAAAAGCCTGGAAAAGCTCCTAAGTTTTTGATTTATTACACTTCCAGGCTGGAGTCCGGTGTCCCATCCAGGTTCAGCGGCAGTGGAAGCGGAACCGACTATACACTGACTATTTCTAGCTTGCAACCCGAGGACTTTGCCACCTATTATTGCCAGCAAGGTAACGCCCTGCCCTGGACCTTCGGTGGCGGAACCAAGGTGGAAATCAAGCGGACAGTGGCCGCACCGTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC 220 <![CDATA[C with A141D H1 > ASTKGPSVFPLAPSSKSTSGGTADLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 221 Substitution for IgG1CH2WT (IMGT) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 222 Substitution for IgG1CH2Fc-null (IMGT) APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Example 2: In vitro assay of LM1486 binding protein The activity of the novel LM1486(CD20) binding protein disclosed in this paper was demonstrated.

[0320] In vitro cell lysis method Assays were performed to measure cell lysis of LM1486 compared to a known CD3 adaptor. Assays were performed for 4 days using the OCI-Ly18 CD20+DLBCL line and PBMCs at a 5:1 E:T ratio. Target B cell viability was assessed by flow cytometry.

[0321] result Figure 1 A and Figure 1 B shows the potent in vitro cell lysis of LM1486 compared to the conventional CD3 adapter. Figure 1 A shows the in vitro cell lysis activity of LM1486 and CD3 adaptors that bind to CD20 monovalently or bivalently. Figure 1 B shows the corresponding EC in the in vitro cell lysis assay. 50 .

[0322] Compared with the activation of CD4 T cells and CD8 T cells with known CD3 adaptors method At the end of the cell lysis assay, T cell activation status was assessed by flow cytometry.

[0323] result Compared to the conventional CD3 adaptor, LM1486 induced a CD8-biased T cell activation profile. Figure 1 C shows the corresponding CD4 T cell activation profile, represented by the percentage of cells expressing CD25 on their cell surface. Figure 1 D shows the corresponding CD8 T cell activation profile, with the percentage of cells expressing CD25 indicated.

[0324] Comparison with cytokine release profiles of known CD3 adaptors method Cytokine release profiles were measured in the supernatant of in vitro cell lysis assays using multiplex assays.

[0325] result Figure 2 A to Figure 2 D shows the in vitro cytokine release profile of LM1486 compared to the conventional CD3 adaptor: IL-6 ( Figure 2 A) IL-10 Figure 2 B), TNF-α Figure 2 C) and IL-17A Figure 2 D). The results show that the LM1486, when used with a CD3xCD20 dual-phase connector ( Figure 26 A) It induces much lower cytokine levels compared to the CD3xCD20 monovalent linker, and the induction level is similar to that of the CD3xCD20 monovalent linker.

[0326] In vitro cell lysis of different CD20+ B cell lines method Different CD20+ B cell lines were incubated with PBMCs at a 5:1 E:T ratio to measure the in vitro cell lysis activity of LM1486. ​​Measurements were performed for 3 to 4 days, and target B cell survival was assessed by flow cytometry.

[0327] result Figure 3 This study demonstrates the potent in vitro cytolytic activity of LM1486 across a range of B cell lines expressing various levels of CD20 antigen density, and also shows the relationship between CD20 density and EC50. 50 There is no correlation between them.

[0328] Comparison of cell lysis activity between LM1486 and CD8 null control and TCR null control method LM1486 was compared with control binding proteins TENG0501 (TCR inactive) and TENG0502 (CD8 inactive). The E:T ratio was determined using the OCI-Ly18 CD20+ DLBCL line and PBMCs at a 5:1 ratio, and target B cell survival and T cell activation profiles were assessed by flow cytometry.

[0329] Figure 4 A to Figure 4 C illustrates the structure of the LM1486 ( Figure 4 A) and the structure of a modified binding protein having a null binding domain that replaces the anti-CD8 domain. Figure 4B) or the structure of the modified binding protein that replaces the ineffective binding domain of the anti-TCR binding domain. Figure 4 C).

[0330] result Figure 4 D shows the in vitro cell lysis activity of LM1486 and the control binding proteins TENG0501 and TENG0502. Figure 4 E shows the corresponding T cell activation profile as assessed by flow cytometry, with the percentage of CD4 and CD8 T cells expressing CD25 shown. The data demonstrate that the LM1486 TCR-binding domain is essential for cytolytic activity, and that the CD8 arm enhances potency and confers CD8-biased T cell activation.

[0331] Comparison of cell lysis activity between LM1486 and a single null binding domain control method LM1486 was compared with control binding proteins TENG0500 (a null binding protein replacing the anti-CD20 domain on the anti-CD8 arm) and TENG0499 (a null binding protein replacing the anti-CD20 domain on the anti-TCR arm). The E:T ratio was determined using the OCI-Ly18 CD20+ DLBCL line and PBMCs at a 5:1 ratio, and target B cell killing and T cell activation profiles were assessed by flow cytometry.

[0332] Figure 5 A to Figure 5 C illustrates the structure of the LM1486 ( Figure 5 A) and the structure of a modified binding protein having a null binding domain that replaces the anti-CD20 domain on the anti-CD8 arm. Figure 5 B) or a modified binding protein structure having a null binding domain that replaces the anti-CD20 binding domain on the anti-TCR arm. Figure 5 C).

[0333] result Figure 5 D shows the in vitro cell lysis activity of LM1486 and control binding proteins TENG0499 and TENG0500. Figure 5 E shows the corresponding T cell activation profile, with the percentage of CD4 and CD8 T cells expressing CD25 shown. These data demonstrate that CD20 binding on the TCR arm is essential for cell lysis.

[0334] Comparison of cell lysis activity between LM1486 and LM1486-2 The difference between LM1486 and LM1486-2 is that LM1486 has RF mutations (see SEQ ID NO: 110), H318R, and Y319F in its TCR heavy chain.

[0335] method B cell survival and T cell activation profiles were assessed by flow cytometry over 3 days using the OCI-Ly18 CD20+DLBCL cell line and PBMCs at an E:T ratio of 5:1.

[0336] result Figure 9 The overlapping in vitro cell lysis spectra of LM1486 and LM1486-2 are shown. Figure 9 A and Figure 9 D shows the cytotoxicity profiles of two different PBMC donors within the T-cell adaptor concentration range. Figure 9 B. Figure 9 C and Figure 9 E, Figure 9 F shows the relevant CD4 and CD8 T cell activation profiles for the two donors, expressed as a percentage of CD25 surface expression.

[0337] Example 3: In vivo efficacy of LM1486 in a B-cell model Evaluation of in vivo antitumor efficacy in a disseminated B-cell model method On day 6, animals were humanized using 5e6 PBMCs. Animals were then treated intraperitoneally twice weekly with PBS or LM1486 at doses of 0.8 mg / kg, 0.08 mg / kg, or 0.008 mg / kg. Tumor burden was monitored twice weekly by bioluminescence imaging, and total throughput (p / s) was recorded.

[0338] result The results showed that the LM1486-binding protein is effective in vivo. Figure 6 Showing the PBS treatment group ( Figure 6 A) at 0.8 mg / kg ( Figure 6 B), 0.08 mg / kg Figure 6 C) and 0.008 mg / kg ( Figure 6 The tumor burden in the LM1486-treated group (D) in the disseminated model. These data demonstrate that LM1486 imparts potent in vivo antitumor efficacy at a low dose of 0.008 mg / kg.

[0339] Compared with known CD3XCD20 connectors, tumor growth inhibition, survival benefit, and superior safety were observed. method 5e6 OCI-Ly18 cells were subcutaneously implanted into the flanks of CD34+ NSG mice (n=8-9 / group). Then, on day 1, the mice were administered 1 mg / kg of a T-cell connective (CD3xCD20 monovalent). Figure 26 B) or a two-way connector ( Figure 26 Animals were treated intraperitoneally with either A) LM1486 or PBS, followed by weekly administration of the connector until day 21. Tumor growth was monitored twice weekly until the end of the study. When the tumor reached 2000 mm... 3 Animals were euthanized at that time. The survival percentage of all treatment groups was reported until the end of the study (day 42), and weight loss over time was monitored as an alternative assessment of safety.

[0340] result Figure 7 This demonstrates the potent tumor growth inhibition, survival benefit, and superior safety of LM1486 relative to known CD3 binders in a fully humanized in vivo model of subcutaneous B-cell tumors. Figure 7 A to Figure 7 C demonstrates the potent tumor growth inhibition, survival benefit, and superior safety of LM1486 compared to conventional CD3 binders in a fully humanized in vivo model of subcutaneous B-cell tumors. Figure 7 C shows the percentage of weight loss recorded 72 hours after the first dose of the T-cell connective.

[0341] Compared with known CD3xCD20 connectors, LM1486 demonstrates antitumor efficacy in an in vivo model of disseminated DLBCL. method On day 0, NSG mice were intravenously injected with the Toledo B cell line expressing luciferase (DLBCL-derived) (Toledo-Luc). On day 7, 10e6 PBMCs were used to humanize mice. On day 21, LM1486 mice were intravenously injected with a single dose of the T-cell connective at 1 mg / kg, while other molecules were injected at equimolar concentrations. On day 24, tumor burden was assessed using bioluminescence imaging (BLI). The total flux (p / s) reflecting tumor burden for each treatment group is shown. Three hours after connective treatment, animals were exsanguinated, and circulating cytokine levels were assessed using multiplex assays.

[0342] result Figure 8 B to Figure 8 E shows the known CD3xCD20 bivalent connector ( Figure 26 A) Compared to TNF-α (pg / mL), cytokine release was reduced (…). Figure 8 B) IL-10 (pg / mL) Figure 8C) IL-2 (pg / mL) Figure 8 D) and IL-17A (pg / mL) Figure 8 E). In addition, Figure 8 F shows a reduction in weight compared to the same known CD3xCD20 bivalent connector. In the in vivo model of disseminated DLBCL, the LM1486 is compared with the known CD3xCD20 bivalent connector ( Figure 26 A) It has a superior safety profile (reduced systemic cytokines and reduced weight loss).

[0343] Example 4: In vitro assay of LRC150016 binding protein A novel T-cell adaptor, LRC150016 (LRRC15), was developed, characterized by two LRRC15-binding domains, one CD8-binding domain, and one TCR-binding domain. Figure 15 ).

[0344] result It combines protein structure and cell-binding activity.

[0345] The LRC150016 and LRC15 were evaluated. Pos and LRRC15 neg Cell line binding. The LRC150016 adaptor demonstrates specific binding with cell lines expressing LRRC15. This is achieved by connecting LRC150016 to LRRC15. pos cell lines ( Figure 10 A) Saos-2WT Figure 10 B) and RPMI WT ( Figure 10 C) LRRC15 neg Cell lines (A431, Saos-2 KO, and RPMI-7951 KO) were incubated at 4°C for 1 hour for assays. LRC150016 was detected using a secondary antibody conjugated with anti-human Fc Alexa Fluor 647 (MFI). LRC150016 is compatible with LRRC15. pos It binds to tumor cell lines, Saos-2, and RPMI7951, but not to LRRC15. neg Cell line A431 binding ( Figure 10 A). When LRRC15 was knocked out from Saos2 WT ​​and RMPI-7951 WT cells, the binding of LRC150016 was lost, demonstrating that the observed binding was specifically attributable to LRRC15 protein binding. Figure 10 B and Figure 10 C).

[0346] LRC150016 mediates T cell effector function in an antigen-dependent manner (LRRC15).

[0347] The effects of LRC150016 on Saos-2 WT (LRRC15) were evaluated. + ) and Saos-2 KO (LRRC15) - In vitro T cell effector function of target cell lines. Purified human T cells were co-cultured with target cell lines at an E:T ratio of 4:1 for 3 days in the presence of LRC150016. The percentage of target cell lysis (cytotoxicity) was measured by CELL-TITRE-GLO. Cytokines (TNFα, IFNγ, and IL-6) were measured in the culture medium collected after 3 days of co-culture using a multiplex cytokine assay. Figure 11 A describes the effect of LRC150016 on Saos-2 WT (LRRC15). + )Target cells but do not induce Saos-2 KO (LRRC15) - The target cells exhibit strong dose-dependent cell lysis. Figure 11 B shows the cytokines associated with the induction of T cell effector function mediated by LRC150016 when T cells are co-cultured with Saos-2 WT cells, but not when co-cultured with LRC15-KO Saos-2 cells. Figure 11 A to Figure 11 B shows that the LRC150016 adaptor mediates T cell effector function in an antigen-dependent (LRRC15) manner.

[0348] LRC150016 specifically induces more CD8 T cells than CD4 T cells targeting LRC15. + Activation of Saos-2 cells.

[0349] To compare LRC150016 with the bispecific LRC15 / CD3 T cell adductor, intact healthy human PBMCs (effectons) were co-cultured with Saos-2 WT (LRRC15+) target cells at an E:T ratio of 4:1 for 4 days in the presence of these molecules. Figure 12 A). After 4 days of co-culture, cell lysis, T cell activation, and secreted cytokines of target cells were measured by CELL-TITRE-GLO, CD25 surface expression assessment by flow cytometry, and multiplex cytokine assays. For both molecules, CD8 T cell activation in this co-culture, as measured by CD25 upregulation, was similar (…). Figure 12 B), but bispecific antibodies targeting CD3 also strongly induce unwanted CD4 T cell activation, which is also shown through CD25 upregulation ( Figure 12 C). Figure 12The study demonstrated that LRC150016 induced potent in vitro cell lysis and CD8-biased T cell activation profiles compared to the comparative CD3 TCE.

[0350] Although the cell lysis activity of the target cells was similar, LRC150016 induced lower levels of cytokines compared to TCE, a comparable drug targeting CD3. Figure 13 A to Figure 13 D). Figure 13 A to Figure 13 D shows the in vitro cytokine profile of LRC150016 compared to the comparative compound CD3 TCE. The cytokine profile was measured in the supernatant of intact PBMCs in an in vitro cell lysis assay using a multiplex cytokine assay. TNFα ( Figure 13 A) IL-6 ( Figure 13 B), IL-10 Figure 13 C) and IL-2 Figure 13 D).

[0351] LRC150016 mediates targeting LRRC15 across a range of LRRC15 expression levels. + T cell lysis activity of cells.

[0352] To understand the correlation between LRRC15 copy number (density) on target cells and LRC150016-mediated cell lysis of target cells, purified human T cells were compared with LRRC15 from various indications. pos The target cell line was co-cultured for 6 days in the presence of LRC150016 at an E:T ratio of 4:1. Cell lysis of the target cells was measured using CELL-TITRE-GLO. From these experiments, E... max and EC 50 The value was calculated and mapped to the corresponding LRRC15 copy number in target cells. In co-cultures of tumor cells and isolated CD3 T cells, LRC150016 induced tumor cell lysis in cells with a more than 10-fold change in LRRC15 antigen copy number. Figure 14 ). Figure 14 A and Figure 14 B demonstrates that LRC150016 exhibits specific in vitro cell lysis activity in a range of cell lines expressing various levels of LRC15.

[0353] Example 5: In vitro and in vivo testing of LM1653 (GPC3) binding protein Combined with kinetic measurements method Binding kinetics were measured using biolayer interferometry on an Octet 384 instrument. The streptavidin (SA) biosensor was loaded with biotinylated protein antigen (ACRO Biosystems, Newark, DE), 1 mg / mL BSA, and 0.05% (v / v) TWEEN (kinetic buffer) in PBS (pH 7.2). The loaded biosensors were washed in the same buffer before association and dissociation measurements at specified times with various antibodies. Kinetic parameters (K0) were calculated using nonlinear fitting of the data using OCTET software v.12.2.1.24. on and K off ) and affinity (K D ).

[0354] Cell binding assay method Cell binding was measured by flow cytometry. Human hepatocellular carcinoma cells HepG2 and Hep3B, human and cynomolgus monkey PBMCs were incubated with serially diluted GPC3 TITAN molecules on ice for 30 minutes, washed with FACS buffer, and stained with goat anti-human IgG-APC (Jackson ImmunoResearch) on ice for 30 minutes before flow cytometry analysis.

[0355] result Figure 25 The diagram shows LM1653 binding to human liver cancer cells and effector cells. Figure 25 Showing GPC3 TITAN LM1653 in EC 50 :8 nM binds to human liver cancer cells Hep3B ( Figure 25 A) and EC 50 :2 nM binds to human liver cancer cells HepG2 ( Figure 25 B). GPC3 TITAN with EC 50 6 nM binds to human PBMCs ( Figure 25 C). Cell binding was measured by flow cytometry. Human hepatocellular carcinoma cells HepG2 and Hep3B, and human PBMCs were incubated with serially diluted GPC3 TITAN molecules on ice for 30 minutes, washed with FACS buffer, and stained with goat anti-human IgG-APC (Jackson ImmunoResearch) on ice for 30 minutes before flow cytometry analysis.

[0356] Table 2. Kinetic analysis of GPC3 protein by anti-GPC3 Fab Table 3. Kinetic analysis of CD8α protein by anti-CD8 VHH Table 2 shows the kinetic analysis of the interaction between anti-GPC3 fab and soluble recombinant human GPC3 protein. Table 3 shows the kinetic analysis of the interaction between anti-CD8 VHH and soluble recombinant human CD8a protein via biofilm interferometry on an OCTET RED384.

[0357] Binding kinetics were measured using biolayer interferometry on an OCTET 384 instrument. The streptavidin (SA) biosensor was loaded with biotinylated protein antigen (ACRO Biosystems, Newark, DE), 1 mg / mL BSA, and 0.05% (v / v) TWEEN (kinetic buffer) in PBS (pH 7.2). The loaded biosensor was washed in the same buffer before association and dissociation measurements at specified times with various antibodies. Kinetic parameters (K0) were calculated using nonlinear fitting of the data using OCTET software v.12.2.1.24. on and K off ) and affinity (K D ).

[0358] Compared to the conventional GPC3xCD3 adaptor, the LM1653-induced in vitro targeting of hepatocellular carcinoma (HCC) cell lines... Cell lysis method GPC3 expression was detected using a commercially available anti-human GPC3 antibody and quantified using the BD Quantibrite kit. Cytotoxicity assays against specified HCC cell lines were performed for 4 days using PBMCs at an E:T ratio of 10:1 using the XCELLIGENCE system.

[0359] result Figure 16 The study demonstrates in vitro cell lysis induced by LM1653 against hepatocellular carcinoma (HCC) cell lines expressing different levels of GPC3, compared to the conventional GPC3xCD3 adapter. Figure 16 A shows GPC3 surface expression across four HCC cell lines. GPC3 expression was detected using a commercially available anti-human GPC3 antibody and quantified using the BD Quantibrite kit. Figure 16 B to Figure 16 E shows LM1653 and monovalent GPC3xCD3 binders targeting the HCC cell line HepG2 ( Figure 16 B), Hep3B Figure 16 C), Huh-7 Figure 16 D) and PLC / PRF / 5 ( Figure 16E) in vitro cell lysis activity. The figure shows the mean ± SEM (n = 3-6 PBMC donors). The tables in the figure show the corresponding EC for each cell line. 50 And lethality E max .

[0360] T cell activation spectrum method T cell activation profiles were determined by flow cytometry. The activation profile was defined as the percentage of parental cells expressing CD25.

[0361] result Figure 17 The diagram shows the CD8-biased T cell activation profile induced by LM1653 against HCC cell lines expressing different levels of GPC3, compared to the conventional GPC3xCD3 adapter. Figure 17 This shows that in the HCC cell line HepG2 ( Figure 17 A), Hep3B Figure 17 B), Huh-7 Figure 17 C) and PLC / PRF / 5 ( Figure 17 Activation profiles of CD8+ T cells (top) and CD4+ T cells (bottom) induced by LM1653 and the monovalent GPC3xCD3 adaptor in cytotoxicity assays (D). The figures show mean ± SEM (n = 3–6 PBMC donors). The tables show ECMO activation for CD8 and CD4 T cells against each cell line. 50 .

[0362] Comparison with cytokine release profiles of known CD3 adaptors method Cytokines released from the supernatant were measured using multiple assays (Luminex).

[0363] result Figure 18 The in vitro cytokine release profile of LM1653 is shown compared to that of the conventional GPC3xCD3 adapter. Figure 18 This demonstrates that in an in vitro cell lysis assay, using HCC Hep3B cell lines as target cells at an E:T ratio of 10:1, the secreted cytokine TNF-α (TNF-α) was... Figure 18 A), (IL-10) Figure 18 B) IL-2 Figure 18 C) and IFN-γ Figure 18 The level of D). Results from a representative PBMC donor from three PBMC donors are shown.

[0364] Compared to the conventional GPC3xCD3 adaptor, LM1653-induced T cell activation and cytokine release profiles were significantly different. method Donor PBMCs (n=3) were incubated with LM1653 and a standard GPC3xCD3 adaptor for 48 hours. CD8 and CD4 T cell activation profiles (defined as the percentage of parental cells expressing CD25) were determined by flow cytometry. Cytokines released in the supernatant at 48 hours were measured using a multiplex assay (LUMINEX).

[0365] result Figure 19 The spectrum of nonspecific T cell activation and cytokine release induced by LM1653 is shown compared to that of the conventional GPC3xCD3 adapter. Figure 19 The nonspecific activity of LM1653 and the conventional GPC3xCD3 adaptor is shown in the plate binding assay. Donor PBMCs (n=3) were incubated with LM1653 and the conventional GPC3xCD3 adaptor for 48 hours. CD8 and CD4 T cell activation profiles (defined as %) of parental cells expressing CD25 were plotted on [data missing]. Figure 19 A and Figure 19 B. Histograms represent mean ± SEM; *, p < 0.05, two-way ANOVA. IL-6 release was measured in the supernatant at 48 hours using multiple determination (LUMINEX). Figure 19 C), TNF-α Figure 19 D), IFN-γ Figure 19 E), IL-2 Figure 19 F), IL-10 Figure 19 G) and IL-17 Figure 19 H). Results from a representative donor PBMC are shown.

[0366] In vitro cell lysis and cytokine release profile induced by LM1653 and conventional GPC3xCD3 adaptor method Using the XCELLIGENCE system, assays were performed for 4 days using purified CD4 or CD8 T cells at an E:T ratio of 5:1. The target cells were HCC GPC3 cells. + Hep3B. Cytokines released from the supernatant were measured using multiplex assay (LUMINEX).

[0367] result Figure 20 The in vitro cell lysis and cytokine release profiles induced by purified CD4 and CD8 T cells, LM1653, and the conventional GPC3xCD3 adaptor are shown. Figure 20The image shows the in vitro cell lysis activity of purified CD4 T cells (top) and purified CD8 T cells (bottom) used in the XCELLIGENCE assay, along with LM1653 and the monovalent GPC3xCD3 adaptor. Figure 20 A to Figure 20 B) and cytokine secretion ( Figure 20 C to Figure 20 J).

[0368] LM1653-induced targeting of GPC3 + In vitro cell lysis of Hep3B method Using the XCELLIGENCE system, dissociated tumor cells from HCC patients were used as the effector cell source in this assay. Tumor-infiltrating T cells were added to the cultures at E:T ratios of 2:1 and 3:1, respectively. The target cells were HCC GPC3+Hep3B cells.

[0369] result Figure 21 This demonstrates the use of tumor-infiltrating lymphocytes derived from HCC patients, induced by LM1653 targeting GPC3. + In vitro cell lysis of Hep3B. Figure 21 Showing from HCC stage II ( Figure 21 A) and Stage IIIB ( Figure 21 B) Patient-derived tumor-infiltrating lymphocytes against GPC3 + LM1653-dependent killing of Hep3B cell lines. Tumor-infiltrating T cells were added to cultures at E:T ratios of 2:1 and 3:1, respectively. The cytotoxic activity of LM1653 and a null control compound that could not bind GPC3 was demonstrated.

[0370] LM1653-induced in vitro cell lysis of GPC3+ lung cancer cells and ovarian cancer cells method Cytotoxicity assays targeting specific ovarian and lung cell lines were performed for 4 days using the XCELLIGENCE system with an E:T ratio of 10:1 using PBMCs.

[0371] result Figure 22 The study demonstrates in vitro cell lysis of GPC3+ lung cancer cells and ovarian cancer cells induced by LM1653, compared to the conventional GPC3xCD3 adapter. Figure 22 The LM1653 and monovalent GPC3xCD3 connector are shown for use in the non-small cell lung cancer line NCI-H661 ( Figure 22 A) and NCI-H2172 ( Figure 22 B) and ovarian cancer cell line OV-90 ( Figure 22 C) and Kuramochi ( Figure 22 D) In ​​vitro cell lysis activity. The table in the figure shows the corresponding EC for each cell line. 50 And lethality E max In vitro cell lysis and T cell activation profile induced by LM1653 and LM1653-2.

[0372] method Cytotoxicity assays for specified HCC cell lines were performed using PBMCs at an E:T ratio of 10:1 in the XCELLIGENCE system for 4 days, with cell lysis activity measured on day 4 (n=1 PBMC donor). T cell activation profiles were determined in the cytotoxicity assays by flow cytometry.

[0373] result Figure 23 The comparable in vitro cell lysis and T cell activation profiles induced by LM1653 and LM1653-2 (which have the same amino acid sequence as LM1653 but were produced in different batches) are shown. Figure 23 A to Figure 23 Table B shows the GPC3+ cell line Hep3B ( Figure 23 A) and Huh-7 Figure 23 B) Corresponding EC 50 And lethality E max Using Hep3B ( Figure 23 C to Figure 23 D) and Huh-7 cell line ( Figure 23 E to Figure 23 F) T cell activation profiles were determined in a cytotoxicity assay. The percentages of CD8 (solid symbol) and CD4 (hollow symbol) T cells expressing CD25 were reported.

[0374] LM1653 for GPC3 高 and GPC3 低 In vivo antitumor activity of HCC xenograft model.

[0375] method GPC3 高 On day 0, NSG mice (n=10 / group) were engrafted with 5e06 Hep3B cells expressing high levels of GPC3. On day 21, the animals were humanized with 10e06 in vitro expanded panT cells. Starting on day 22, the animals were treated weekly with PBS, LM1653 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, and 0.03 mg / kg) and conventional GPC3xCD3 connector (1 mg / kg and 0.1 mg / kg) via intraperitoneal injection for a total of 4 injections. Tumor burden was measured over time and reported as mean ± SEM. ‡ 40% were tumor-free; **, p<0.01 and ***, p<0.001, two-way ANOVA ( Figure 24 A).

[0376] GPC3 低 On day 0, NSG mice (n=10 / group) were engrafted with 5e06 PLC / PRF / 5 cells expressing low levels of GPC3. On day 5, the animals were humanized with 10e06 in vitro expanded panT cells. Starting from day 6, the animals were treated twice weekly with PBS and LM1653 (3 mg / kg, 1 mg / kg, and 0.3 mg / kg) via intraperitoneal injection for a total of 7 injections. Tumor burden was measured over time and reported as mean ± SEM. **, p<0.0001, two-way ANOVA (… Figure 24 B).

[0377] result Figure 24 The LM1653 is shown to be designed for GPC3. 高 and GPC3 低 In vivo antitumor efficacy of HCC xenograft model. Dashed line indicates TCE administration.

[0378] In vivo antitumor activity and related cytokine secretion of LM1653 in mice carrying Hep3B tumors method NSG mice were irradiated with 200 cGy on day -3 (n=11-12 per treatment group) and implanted with 7×10⁻⁶ cells via IP on day 0. 6 One Hep3B-luciferase cell was implanted into mice via IV fluid on day 8. 1 × 10⁶ cells from a healthy donor were then transplanted into the mice via IV fluid. 7 Individual PBMCs. On day 22, mice were injected intraperitoneally with either PBS, LM1653-2, or GPC3xCD3 at a dose of 1 mg / kg. Tumor burden was monitored twice weekly by bioluminescence imaging (BLI) until day 24, two days post-treatment.

[0379] result Figure 41 The results showed that, compared with the conventional GPC3xCD3 connector, LM1653 induced significant tumor growth inhibition in mice carrying Hep3B tumors, without significant secretion of related cytokines.

[0380] Figure 41 A shows the tumor growth inhibition induced by both LM1653-2 and the conventional GPC3xCD3 connector. Mean + / - SEM tumor burden across experimental cycles is shown for each treatment group. Dashed lines indicate treatment days. Statistical significance of differences in tumor burden over time was determined using two-way ANOVA with Tukey's multiple comparison test (p < 0.0001; ns = not significant).

[0381] exist Figure 41 B to Figure 41 In E, serum cytokine concentrations were assessed by multiplex assays at 6 hours post-TCE administration. IFN-γ (IFN-γ) levels were plotted for each treatment group. Figure 41 B), TNF-α Figure 41 C), IL-10 Figure 41 D) and IL-2 ( Figure 41 Individual values ​​of E) and mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were used to determine the statistical significance of the differences.

[0382] In vivo tumor growth inhibitory activity and related cells of LM1653-3 in mice carrying NCI-H661 lung tumors Factor secretion method NSG mice were irradiated with 200 cGy on day -3 (n=5-7 per treatment group) and implanted intravenously with 2.5×10⁻⁶ styrene tablets on day 0. 6 One NCI-H661-luciferase cell was implanted into the mouse IV cell line on day 12. 1 × 10⁶ cells from a healthy donor were then transplanted into the mouse IV cell line. 7 Individual PBMCs. Starting on day 27, mice were injected 2QW IV with either the solvent (PBS) or LM1653-3 at 1 mg / kg and 0.3 mg / kg, for a total of 4 doses. OKT3 was used as a positive control for T cell activation and cytokine secretion. LM1653-3 has the same amino acid sequence as LM1653 but was produced in different batches.

[0383] result Figure 42 A shows that both doses of LM1653-3 induce tumor growth inhibition.

[0384] Figure 42This study demonstrates that LM1653-3 induced significant tumor growth inhibition in mice carrying NCI-H661 lung tumors compared to solvent-treated animals, without significant associated cytokine secretion. Tumor burden was monitored twice weekly by bioluminescence imaging (BLI). The geometric mean + / - SD of tumor burden across experimental cycles is shown in each treatment group. Dashed lines indicate treatment days. Statistical significance of differences in tumor burden over time was determined using two-way ANOVA with Tukey's multiple comparison test (*, p < 0.05; ** p < 0.01; **** p < 0.0001). LM1653-3 exhibited the same in vitro bioactivity profile as LM1653 and LM1653-2.

[0385] exist Figure 42 B to Figure 42 In E, serum cytokine concentrations were assessed by multiplex assays at 3 hours after the first TCE or OKT3 administration. IFN-γ (IFN-γ) levels were plotted for each treatment group. Figure 42 B), TNF-α Figure 42 C), IL-10 Figure 42 D) and IL-2 ( Figure 42 Individual values ​​of E and mean ± SEM.

[0386] Example 6: Efficacy of LM1653 (GPC3 TITAN) in a cynomolgus monkey model method Kinetic measurements of the soluble forms of GPC3 and CD8ab were performed using a BIACORE instrument. D k is calculated as a nonlinear fit from the data. off / k on Ratio. Values ​​are presented as the mean ± standard deviation (SD) of three experiments. Due to measurement limitations, the kinetics of TCR binding cannot be accurately determined.

[0387] PBMCs from three different healthy human or four cynomolgus monkey donors were incubated with HCC GPC3+ HepG2 at a 10:1 effector-to-target ratio. LM1653 was added in 5-fold serial dilutions starting at 50 nM, for a total of nine dilutions. The percentage of cell lysis was assessed 48 hours after PBMC addition using an XCELLIGENCE impedance-based RTCA MP analyzer.

[0388] LM1653 was administered to cynomolgus monkeys (n=3 per treatment group and each sex) via intravenous (bolus) or subcutaneous injection weekly at doses of 1 mg / kg / week IV, 6 mg / kg / week IV, or 6 mg / kg / week SC for 1 month (5 doses), followed by a 2-week recovery period. Animals that received only the solvent in either IV or SC form served as controls.

[0389] result Table 4 shows the binding kinetics of LM1653 with recombinant human and cynomolgus monkey GPC3 and CD8 proteins.

[0390] Table 4. Table 5 shows the LM1653-induced T cell-dependent cytotoxicity (TDCC) in human and cynomolgus monkey PBMCs and the HCC HepG2 cell line. Table 5 also shows the TDCC EC5 values ​​for both human and cynomolgus monkey PBMCs. 50 and E max Median and interquartile range (IQR).

[0391] Table 5. Figure 43 The effects of LM1653 administration in cynomolgus monkeys are shown. Figure 43 A shows the absolute number of CD8 T cells in the blood during treatment. Administration of LM1653 was associated with a transient decrease in the number of CD8 T cells in the blood, a result of T cell lateralization. Lateralization is considered an expected effect of LM1653 activity as a result of the drug's interaction with target T cells.

[0392] Figure 43 B to Figure 43 E shows serum cytokine and chemokine levels after the first dose. LM1653 was associated with a transient increase in chemokines MCP-1 and MIP-1b, which are associated with CD8 T cell migration and function. For the CRS-associated cytokines IL-10 and IL-6, only small and transient increases were observed, consistent with preclinical data generated in vitro and in mice. LM1653 activity was only accompanied by a minimal increase in circulating cytokine levels, potentially reducing the risk of CRS-related toxicity in patients. Solid and dashed lines represent values ​​obtained for males and females, and for solvent controls, respectively.

[0393] Example 7: Humanization of TCR VHH framework residues The anti-TCR VHH amino acid sequence of SEQ ID NO: 42 has camelidoidal properties. Amino acid residues were humanized to minimize immunogenicity, with non-vernier frame residues altered to match the closest human lineage sequence IGHV3-23*1. During humanization, the nanobody marker residues Y42 and K52 in frame 2 were not altered. A lead humanized VHH group was generated, with marker residues E49 and R50 either humanized or non-humanized. Approximately 40 humanized variants were evaluated using a 90% binding retention threshold set. Binding retention of the humanized clones was tested by generation as Fc fusions prior to introduction as trispecific antibodies, followed by cell binding assays of binding kinetics. Mutations from the variants that performed best in the Fc fusions were combined to generate six humanized anti-TCR VHHs containing 5 to 10 humanized residues. These were integrated into the final trispecific form. Cytotoxicity and T cell activation of the final lead group were evaluated. All variants met criteria in the final form. Select the following TCR V HH : hu2.1 (SEQ ID NO: 43), hu2.2 (SEQ ID NO: 44), hu2.3 (SEQ ID NO: 45), hu2.4 (SEQ ID NO: 46), hu2.5 (SEQ ID NO: 47) and hu2.6 (SEQ ID NO: 48).

[0394] Example 8: Efficacy of LM1486 (CD20 TITAN) in models of inflammatory diseases and autoimmune disorders method Human peripheral blood mononuclear cells (PBMCs) Primary human PBMCs were isolated from whole blood obtained from patients with SLE or myositis. PBMCs were counted, and 100 kJ / well of PBMC was treated with different concentrations of CD20 TITAN molecules in 96-well plates. PBMCs were cultured aseptically in a CO2 incubator at 37°C for three days. At the end of the three-day period, B cell exhaustion was measured by flow cytometry, and IC50 was calculated using PrismGraphPad software. 50 value.

[0395] Humanized mouse model Figure 29 The method steps are described. NOD-scid IL2Rγ 无效(NSG) mice were obtained from the Jackson laboratory approximately 22 weeks post-transplantation, randomized, and treated via intraperitoneal (ip) injection with either solvent (PBS), a 1 mg / kg NIP228 control, or 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg LM1486. ​​Blood and tissues were collected 72 hours post-treatment and processed for further analysis.

[0396] Using a syringe plunger, pass the spleen along with 5 mL of neutralization buffer RPMI 1640 (GIBCO) containing 5% FBS (GIBCO) and 2 mM EDTA (INVITROGEN) through a cell filter and centrifuge at 460 g for 5 minutes at 4°C. Resuspend the cell pellet in 1 mL of ACK lysis buffer (GIBCO) for 1 minute, then add 2 mL of neutralization buffer, centrifuge at 460 g for 5 minutes at 4°C, and then resuspend the pellet in 2 mL of neutralization buffer.

[0397] Using a syringe plunger, thymus cells were passed through a cell filter along with 5 mL of neutralization buffer and centrifuged at 460 g for 5 minutes at 4°C. The cell pellet was then resuspended in 2 mL of neutralization buffer.

[0398] Bone marrow cells were flushed from the femur and tibia of the hind leg using a 25G needle and passed through a cell filter to remove any bone spurs, muscle or cell clumps. Red blood cells were lysed with ACK lysis buffer (GIBCO). After centrifugation, the pellet was resuspended in cold neutralization buffer.

[0399] Blood samples were collected in EDTA pretreated tubes (BD) and centrifuged at 500g for 10 minutes at 4°C to separate plasma from cells. The cell pellet was resuspended in 5 mL of ACK lysis buffer (GIBCO) at room temperature for 90 seconds to lyse red blood cells. The cells were washed with 5 mL of neutralization buffer and resuspended in 1 mL of cold neutralization buffer.

[0400] The capsule and any extra connective tissue were removed from all kidney samples using tweezers and gently crushed with a syringe plunger. This was done using Miltenyi gentleMACS. ™ The dissociation scheme achieves dissociation.

[0401] In summary, samples were collected into Miltenyi GentleMACS C tubes containing 2 mL of multi-tissue dissociation medium and incubated at 37°C for 30 min on program “37 Multi E”. The cell suspension was neutralized by adding 2 mL of neutralization buffer and filtered through a 40 μm cell filter. After centrifugation, the pellet was resuspended in 40% Percoll (GE HEALTHCARE) and centrifuged at 580 g for 20 min at room temperature. Cells at the bottom were collected, washed with neutralization buffer, and then resuspended in 1 mL of cold neutralization buffer. For flow cytometry analysis: 2 million cells were mixed with live / dead NIR 780 (THERMOFISHER) diluted 1:2000 in 200 μl of a solution containing 2 μg of Fc inhibitor. Incubate together in RPMI 1640 (BIOLEGEND) at 4°C for 5 minutes. Wash cells with FACS buffer by centrifugation at 460g for 5 minutes at 4°C. The precipitate was resuspended in 50 μl of an antibody mixture containing 2.5 μl of Superbright staining buffer for staining and incubated on ice for 30 min. The antibody mixture contained anti-human CD3 BUV 395 (BD, 1:100 dilution), anti-human CD19 BUV737 (BD, 1:200 dilution), anti-human CD8a BV711 (BIOLEGEND, 1:400 dilution), anti-human CD2 BB515 (BD, 1:1200 dilution), anti-human CD25 PE (BIOLEGEND, 1:50 dilution), anti-human CD4 PE-Cy7 (BIOLEGEND, 1:400 dilution), anti-human CD45 APC (BIOLEGEND, 1:200), and anti-mouse CD45 BV510 (BIOLEGEND, 1:400 dilution). Cells were washed twice with FACS buffer and then fixed for 20 min at room temperature by adding 100 μl of 1X fixation buffer (EBIOSCIENCE). After centrifugation, the cells were resuspended in 200 μl of FACS buffer. Flow cytometry was performed using a Symphony A3 (BD ​​BIOSCIENCE) analyzer. Analysis was performed using FLOJO software.

[0402] Lymph nodes were fixed in 4% PFA for 48 hours, dehydrated, and embedded in paraffin blocks. Four-micrometer-thick tissue sections were cut and stained with an immunofluorescence kit (AKOYA BIOSCIENCES) containing OPAL dyes for detection of: anti-CD19 (EPR5906, Abcam; OPAL 520), anti-CD25 (EPR6452, Abcam; OPAL 570), anti-CD8 (D8A8Y, CellSignaling Technology; OPAL 620), and anti-CD4 (EPR6855, Abcam; OPAL 480). Digital whole-section images were generated using PhenoImagerHT (AKOYA BIOSCIENCES), and cell classification and tissue area quantification were performed using VISIOPHARM v.2023.09.5.15777.

[0403] result IC50 of B-cell depletion and maximum B-cell depletion in PBMCs from SLE and myositis patients 50 The calculations of the values ​​are depicted in Table 6.

[0404] Table 6. B-cell depletion and maximum B-cell depletion in SLE and myositis Patient ID <![CDATA[B cell exhaustion IC 50 (pM)]]> % of maximum B cell exhaustion Myo 109785 107.5 96 Myo 107370 2.8 94 Myo 120323 7.1 62 Myo 109852 1.1 86 Myo 112243 3.8 89 Myo 123136 13.3 68 Myo 138502 38.1 84 SLE 29144 8.5 81 SLE 89313 1.2 88 SLE 20643 11.5 60 SLE 23199 1.4 27 SLE 91744 19.1 41 SLE 23150 0.5 63 SLE 29942 0.3 41 SLE 29941 1.8 75 Figure 27 and Figure 28 The efficacy and safety of LM1486 (CD20 TITAN) were demonstrated. LM1486 showed excellent and potent B-cell depletion in cells derived from SLE and myositis patients. Figure 27 A and Figure 27 B), while also demonstrating excellent safety and selective CD8+ T cell activation (B). Figure 27 E and Figure 27 F) and CD4 T cell retention activity Figure 27 C and Figure 27 D). Figure 28 A and Figure 28 B shows IL-17A in nonspecific T cell activation assays ( Figure 28 A) and TNFα Figure 28 B) Reduced release of cytokines.

[0405] By using flow cytometry, LM1486 (CD20 TITAN) achieved deep B cell depletion in blood and tissue compartments of humanized mouse models 72 hours after a single dose treatment in NSG mice transplanted with human CD34+ cells. Figure 30 A to Figure 30 F depicts the spleen in a humanized mouse model following a single dose of LM1486 (CD20 TITAN). Figure 30 A) Blood ( Figure 30 B) Bone marrow Figure 30 C) Kidney ( Figure 30 D) Thymus ( Figure 30 E) and lymph nodes ( Figure 30 B cell depletion was observed in F). Depletion was more pronounced in peripheral blood and spleen.

[0406] Example 9: Efficacy of LM1486 (CD20 TITAN) in a cynomolgus monkey model method Crab-eating macaques were administered either a solvent (n=10) or 3 mg / kg of CD20 TITAN twice weekly (n=10). The CD20 TITAN group received a total of 5 doses, and an autopsy was performed on day 16. Figure 44 Mean ± SEM B cell counts (CD19+) were assessed by flow cytometry, and peripheral blood (day -14 to day 16) and tissue (day 16) counts were presented. Absolute circulating CD8+ and CD4+ T cells and the fold change in activated CD8 / CD4 (CD25+) relative to baseline were assessed during the first week of treatment following the first dose. Finally, the group mean plasma cytokine profile (IL-6, TNFα, and IFNγ) was also assessed over the time course from day -14 to day 16.

[0407] result In the cynomolgus monkey model, LM1486 (CD20 TITAN) at 3 mg / kg IV exhibited potent B-cell depletion activity with low systemic cytokine release and associated toxicity. Figure 45 The image shows circulating CD19+ B cells in the spleen, bone marrow, and lymph nodes. Figure 45 A) and tissue-specific CD19+ B cells ( Figure 45 B) depletion. Figure 46 This shows T cell attachment and preferential CD8 activation. Absolute circulating CD8+ (CD8+) after the first dose. Figure 46 A) and CD4+ T( Figure 46 B) Cells and activated CD8 / CD4 (CD25+) Figure 46 C) shows the multiple difference relative to baseline during the first week of treatment. Figure 47 The diagram shows the timeline from day -14 to day 16, for IL-6 ( Figure 47 A), TNFα Figure 47 B) and IFNγ Figure 47 Group C) mean plasma cytokine profile.

[0408] Example 10: In vitro and in vivo testing of LM1953 (STEAP2 TITAN) This paper presents the LM1953 (STEAP2, see [link]). Figure 43 D) The activity of binding proteins.

[0409] In vitro cell lysis, T cell activation and cytokine release profile method Assays were performed to measure the cytotoxic activity of LM1953. In the XCELLIGENCE assay, STEAP2+ prostate cancer lines 22Rv1 and C4-2, along with PBMCs from eight donors, were measured over 3 days at an E:T ratio of 10:1. At the end of the cell lysis assay, T cell activation status was assessed by flow cytometry, and the cytokine release profile in the supernatant was measured using multiplex assays.

[0410] result Figure 31 A and Figure 31 B shows the potent in vitro cell lysis of LM1953. Figure 31 A shows the in vitro cell lysis activity of LM1953 against C4-2 cells and the corresponding EC50. 50 . Figure 31 B shows the in vitro cell lysis activity of LM1953 on 22Rv1 cells and the corresponding EC50. 50 LM1953 induced a CD8-biased T cell activation profile. Figure 32 A shows the results in the C4-2 cell lysis assay and Figure 32 B shows the corresponding CD4 and CD8 T cell activation profiles in the 22Rv1 cell lysis assay, indicated by the percentage of cells expressing CD25 on the cell surface. Figure 33 A to Figure 33 D shows the effect of LM1953 on IL-6 in C4-2 cells ( Figure 33 A) and TNF-α Figure 33 B), and on 22Rv1 cells, IL-6 ( Figure 33 C) and TNF-α Figure 33 D) In ​​vitro cytokine release profile.

[0411] Antitumor efficacy of LM1953 in humanized prostate cancer xenograft cell line models method On day -7, humanized male NSG MHC I / IIDKO mice (n=4 / group) were intravenously injected with 10e6 PBMCs from 3 different donors, and then 5e6 22Rv1 tumor cells were subcutaneously injected into the flank on day 0. On days 2, 6, 10, 14, 17 and 21, LM1953 was injected intraperitoneally at 0.41 mg / kg, and tumor size was assessed using calipers.

[0412] result Figure 34 This demonstrates the in vivo antitumor efficacy of LM1953 against the STEAP2+ prostate cancer xenograft model 22Rv1.

[0413] Example 11: LRC150016 (LRRC15) Fab variant with improved exploitability spectrum Design LRC150016 V H / V L Sequence variants were developed to improve the developability and humanization of LRRC15 Fab. These variants were generated as antibodies in the form of human IgG1 using standard methods and tested for retention of binding to recombinant LRRC15 protein in humans, mice, and cynomolgus monkeys. Notably, variants with more human germline residues within H-CDR2 (SEQ ID NO: 136, 147, 152, and 157) were found to retain binding to LRRC15.

[0414] The selected LRRC15-binding Fab variant (see Table 7 below) is then used to form a T-cell adaptor in the form of CD8 / TCR (see Table 7 below). Figure 15 It has two LRRC15-bonded Fab arms and one TCR-bonded V. HH Combined with 1 CD8 V HH The CD8 binding sequences were replaced with variants modified to remove two sequence susceptibility sites identified in the original variants. One of these changes was in CDR1 as defined by IGMT. The ability of these T cell adaptors to induce CD8-mediated cell lysis in tumor cell lines expressing LRRC15 in co-culture with human PBMCs was tested.

[0415] Table 7. Candidate Number LRRC15 Fab VH LRRC15 Fab VL TCR VHH CD8 VHH LRC150016 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 42 SEQ ID NO: 52 TPP-46956 SEQ ID NO: 141 SEQ ID NO: 142 SEQ ID NO: 42 SEQ ID NO: 168 TPP-46957 SEQ ID NO: 148 SEQ ID NO: 142 SEQ ID NO: 42 SEQ ID NO: 168 TPP-46958 SEQ ID NO: 153 SEQ ID NO: 142 SEQ ID NO: 42 SEQ ID NO: 168 TPP-46959 SEQ ID NO: 159 SEQ ID NO: 160 SEQ ID NO: 42 SEQ ID NO: 168 TPP-46960 SEQ ID NO: 159 SEQ ID NO: 166 SEQ ID NO: 42 SEQ ID NO: 168 TPP-47826 SEQ ID NO: 153 SEQ ID NO: 142 SEQ ID NO: 172 SEQ ID NO: 168 TPP-49058 SEQ ID NO: 159 SEQ ID NO: 160 SEQ ID NO: 172 SEQ ID NO: 168 The affinity of T cell adaptors for recombinant human, mouse, and cynomolgus monkey LRRC15 proteins was measured using the single-cycle kinetics (SCK) method via surface plasmon resonance on the Biacore 8K+ system (CYTIVA).

[0416] The unwanted binding of the T-cell adaptor to cells transiently transfected with human EphB6 was also tested. TPP-46959 was identified as having reduced binding to cells expressing EphB6. This molecule differs from TPP-46960 at L-CDR2 (SEQ ID NO: 158), where residue 55 in TPP-46960 is E55, compared to H55 in huM25 and N55 in TPP-46959.

[0417] Affinity determination of LRRC15(huM25) Fab sequence variant - single-cycle kinetic measurements .

[0418] T-cell adaptors were captured on a PROTEIN G chip and exposed to a series of increasing concentrations of human, mouse, or cynomolgus monkey LRRC15 recombinant extracellular domain protein (Acro Biosystems PROTEIN G). This was achieved using Biacore. ™ The Insight evaluation software (CYTIVA) globally fits the fully calibrated combined data to a 1:1 combined model to determine the KD. Colored sensor traces represent the original data, and black lines represent a global 1:1 fit to that data. The kinetic parameter values ​​shown in Table 8 are the averages of 3 or 4 repeated measurement sets.

[0419] Table 8. Functional evaluation of LRRC15(huM25) Fab sequence variant .

[0420] Complete PBMC (3 donors) with LRRC15 pos Saos-2 Wt target cell lines were cultured for 3 days at an E:T ratio of 10:1. LRC150016 and its LRRC15 Fab sequence variants mediated T cell activation, as measured by % CD25 surface expression. Tabular data (Table 9 below) refer to the TCE molecules EC of CD8 T cell activation. 50 (pM) and E max (%)value.

[0421] Table 9. TCE <![CDATA[EC 50 (pM)]]> <![CDATA[E max (%)]]> LRC150016 3.3 52.31 TPP-46956 3.9 53.32 TPP-46957 3.7 49.69 TPP-46958 3.2 55.17 TPP-46859 2.0 54.63 TPP-46960 2.8 55.06 The results are shown in Figure 35 In A, the value is the mean (±SEM) frequency of T cell activation.

[0422] Dose-response curves demonstrate that, within intact PBMCs and LRRC15 pos After 3 days of co-culturing Saos-2 Wt target cells, both LRC150016 and LRC15 Fab sequence variants of TCE mediated T cell activation in a dose-dependent manner, as measured by CD25 surface expression. Similar to LRC150016, all five LRC15 Fab sequence variants of TCE selectively activated CD8+. + T cells, while not showing CD4 + T cell activation. The values ​​shown here are CD25 values ​​in CD8 and CD4 T cells. + Mean (±SEM) frequency of cells. n=3 donors from representative experiments.

[0423] LRC150016 and its LRRC15 Fab sequence variants target LRRC15 pos In vitro cell lines of Saos-2 WT target cell line Cell lysis activity Target cell lysis was assessed using CELL-TITER-GLO. The tabular data refers to the TCE molecules (ECs) in the target cells during lysis. 50 (pM) and E max (%) values, see Table 10.

[0424] The results are shown in Figure 35 In B, the dose-response curves show that, compared to LRC150016, after 3 days of co-culturing Saos-2 target cells with intact PBMC effector cells, all five LRC15 Fab sequence variants mediated similar or slightly higher levels of TCE-mediated cell lysis of target (Saos-2 Wt) cells, as shown by cell lysis E. max Values ​​were measured. Cell lysis of target cells was assessed using CELL-TITER-GLO. Nip 228 control TCE did not mediate any cytotoxicity of target cells. The values ​​shown here are the mean (±SEM) frequency of cell lysis (cytotoxicity) of target cells. n=3 donors from representative experiments.

[0425] Table 10. TCE <![CDATA[EC 50 (pM)]]> <![CDATA[E max (%)]]> LRC150016 6.7 46.84 TPP-46956 8.7 59.65 TPP-46957 5.3 49.82 TPP-46958 6.1 57.72 TPP-46859 4.4 60.02 TPP-46960 4.3 60.57 Assess the cytokine (IL-6) profile in the supernatant of intact PBMC co-culture assays. .

[0426] In the presence of LRC150016 or one of the five LRC15 fab sequence variants TCE, the complete PBMC was compared with LRC15 at an ET ratio of 10:1. pos Saos-2 Wt target cells were co-cultured for 3 days. The surrogate IL-6 for cytokine release syndrome (CRS) was measured in the culture supernatant after 3 days of co-culture using a standard ELISA. The values ​​shown here are the mean (±SEM) frequency of IL-6 (pg / mL) in the cell culture supernatant. Results are presented in… Figure 35 In C, the LRRC15 variant has a similar effect on IL-6 levels as LRC150016.

[0427] EphB6 Off-target Binding Evaluation Ad293 cells were transiently transfected with a plasmid expressing human EphB6. The binding of the LRRC15 T cell adjuvant was assessed by flow cytometry.

[0428] The results are shown in Figure 36 In comparison with other sequence variants, TPP-46959 showed reduced off-target binding with AD293 cells expressing EphB6.

[0429] Example 12: Plasmablastocyte / plasma cell depletion in primary assay using TPP-42197 (BCMA TITAN) method Healthy donor PBMCs were thawed, counted, and naïve B cells were isolated using magnetic beads. Cells were cultured for 5 days in ATCC-rich RPMI medium with 1 μg / ml CpG and 100 U / ml IL-2 to induce B cell differentiation into plasmablasts. After 5 days of differentiation, autologous PBMCs were thawed and co-cultured with differentiated plasmablasts at a target cell to effector cell ratio of 1:5. To maintain this ratio, the percentage of CD3+ T cells per donor was assessed by flow cytometry, and the number of PBMCs to be cultured was determined, thus maintaining this ratio of plasmablasts to T cells (50,000 plasmablasts to 250,000 CD3+ T cells). Cells were stimulated for 48 hours with increasing concentrations (0.0000003 nM to 20 nM) of TPP-42197 or the comparative drug "comparative T" (CD3xBCMA TCE). At the end of the experiment, the supernatant was collected, and cytokine secretion levels from the cells were assessed by MSD ELISA. The cell lysis level of plasmablasts and the T cell activation levels of CD8+ cytotoxic T cells and CD4+ helper T cells were measured by flow cytometry.

[0430] result TPP-42197 was found to have better potency and better EC than the comparative substance T. 50 (2.2pM vs. 415pM respectively), see [link / reference] Figure 38 Furthermore, TPP-42197 induces selective activation of CD8+ T cells but not CD4+ T cells, which are major contributors to cytokine secretion. Figure 39 A to Figure 39 B). Conversely, the comparative agent T induced activation of both CD8+ and CD4+ T cells. TPP-42197 demonstrated a superior cytokine profile compared to the comparative agent T, exhibiting overall lower cytokine secretion, particularly those involved in cytokine release syndrome (CRS) events (see [link to TPP-42197]). Figure 40 A to Figure 40 F) TCP-42197 completely depleted bone marrow and spleen transplanted human BCMA+ cells To evaluate the ability of TCP-42197 to deplete primary B cells expressing BCMA in vivo, a pharmacodynamic (PD) study (XenoGvHD) was conducted in an allogeneic model of graft-versus-host disease. To prepare for this XenoGvHD PD study, fresh Leukopak (StemExpress) cells were used. ™ Healthy human donor peripheral blood mononuclear cells (PBMCs) are isolated from the cell and cryopreserved (in liquid nitrogen tanks) until transplantation.

[0431] On day -1, 8–10-week-old female NOD scid-gamma mice (NSG; Jackson lab) were pretreated with sublethal whole-body irradiation (1 Gy). On day 0, 15 million total PBMCs from healthy human donors were intravenously injected. On day 9, mice received intraperitoneal administration of phosphate-buffered saline (PBS), 1 mg / kg TCP-42197, 10 mg / kg TCP-42197, or 10 mg / kg isotype control (n=5–9 mice / group). On day 12, mice were euthanized and tissues were collected for FACS analysis to determine whether these TCEs depleted BCMA+ cells present in t...

Claims

1. A binding protein comprising four polypeptide chains forming two tumor-associated antigen (TAA) binding sites against phosphatidylinositol proteoglycan-3 (GPC3), a T-cell receptor binding site, and a T-cell co-stimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the following formula: In L -C L The third polypeptide chain has a structure represented by the following formula: In H1 -C H1 -V HHa -Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: In H1 -C H1 -V HHb -Fc b The first polypeptide and the third polypeptide form the first TAA binding site of the two TAA binding sites, and the second polypeptide and the fourth polypeptide form the second TAA binding site of the two TAA binding sites. in: V L It is the variable domain of the immunoglobulin light chain, and V H1 These are variable domains of the immunoglobulin heavy chain, which together form a TAA-binding domain that specifically binds to tumor-associated antigens; C L It is the constant structural domain of the immunoglobulin light chain; C H1 It is the CH1 heavy chain constant domain of immunoglobulin; V HHa It is a single-stranded variable domain that specifically binds to the T-cell receptor; V HHb It is a single-stranded variable domain that specifically binds to T cell co-stimulatory molecules; Fc a It is C H2a and C H3a Immunoglobulin heavy chain constant domain; and Fc b It is C H2b and C H3b Immunoglobulin heavy chain constant domain.

2. The binding protein according to claim 1, further comprising: L1, namely, C located on the third polypeptide chain. H1 With V HHa The linker between; and L2, namely V located on the third polypeptide chain. HHa With the Fc a The joints between L1 and L2, where L1 and L2 are either joints or do not exist independently.

3. The binding protein according to claim 1 or claim 2, wherein the binding protein further comprises: L3, namely, C located on the fourth polypeptide chain. H1 With V HHb The linker between; and L4, namely the V located on the fourth polypeptide chain. HHb With Fc b The joints between L3 and L4 are either independent joints or do not exist.

4. The binding protein according to any one of claims 1 to 3, wherein the binding protein further comprises: H1, namely, C located on the third polypeptide chain. H1 With V HHa The immunoglobulin hinge region between; and H2, i.e., V located on the third polypeptide chain. HHa With the Fc a The immunoglobulin hinge regions between H1 and H2, where H1 and H2 are either independently immunoglobulin hinge regions or do not exist.

5. The binding protein according to claim 4, wherein H1 comprises SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 90 or is absent, and wherein H2 comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 90 or is absent.

6. The binding protein according to any one of claims 1 to 5, wherein the binding protein further comprises: H3, namely, C3 located on the fourth polypeptide chain. H1 With V HHb The immunoglobulin hinge region between; and H4, namely V located on the fourth polypeptide chain. HHb With the Fc b The immunoglobulin hinge regions between H3 and H4, where H3 and H4 are either independently immunoglobulin hinge regions or do not exist.

7. The binding protein according to claim 6, wherein H3 comprises SEQ ID NO: 53, DK (SEQ ID NO: 54), SEQ ID NO: 90 or is absent, and wherein H4 comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 91 or is absent.

8. The binding protein according to any one of claims 1 to 7, wherein the first polypeptide chain and the second polypeptide chain have a structure represented by the following formula: In L -C L The third polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H1-L1-V HHa -H2-L2-Fc a ,or In H1 -C H1 -H1-L1-V HHa -L2-H2-Fc a Furthermore, the fourth polypeptide chain has a structure represented by the following formula: V H1 -C H1 -H3-L3-V HHb -H4-L4-Fc b ,or In H1 -C H1 -H3-L3-V HHb -L4-H4-Fc b 。 9. The binding protein according to any one of claims 1 to 8, wherein Fc a and / or Fc b From IgG antibodies.

10. The binding protein of claim 9, wherein the Fc a and / or the Fc b Derived from IgG1 antibody.

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