Treatment of myeloid disorders and acute leukemia by targeting novel tumor-specific antigens - Patent Application 20070123333

Abstract

The present disclosure relates to a method for diagnosing or treating myeloid disorders and acute leukemia by using tumor-specific antigens selected from CD63, CD151, CD72, CD84, CD69 and CD109.Furthermore, antigen binding protein (ABP), ABP-drug conjugate, and CAR targeting tumor-specific antigen, and the method for their use are provided.The present disclosure provides a method for diagnosing myeloid disorders (MD) and acute leukemia (AL) in a subject, comprising detecting the presence or level of tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 327,757, filed April 5, 2022, and U.S. Provisional Patent Application No. 63 / 478,068, filed December 30, 2022, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] 1. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on April 5, 2023 is named 34619-55205_WO(005WO) and is 1,66998 bytes in size. [Background technology]

[0003] 2.Background Acute myeloid leukemia (AML) is a hematological cancer that originates in the bone marrow and accounts for approximately one-third of all childhood malignancies. AML causes the largest number of cancer-related deaths in children, with overall survival ranging from 55-70% at 5-year follow-up. Chemotherapy has been the standard AML treatment for more than 40 years, but it often puts the cancer into remission and rarely completely eliminates the cancer cells. This often leads to disease recurrence and ultimately the death of the patient, as second-line treatments are limited. Aggressive post-remission treatments, such as high-dose chemotherapy and hematopoietic cell transplantation, are currently employed in more than 50% of patients after achieving an initial remission. There are no available treatment options for many relapsed patients who are not healthy enough to tolerate such aggressive post-remission treatments.

[0004] Thus, great efforts have been made to develop alternative first-line and post-remission treatments for AML, but without notable success. AML cancer cells show variations in transcription factor occupancy and transcription regulation, and AML patients have various subtypes of leukemia-related symptomatic and prognostic differences. It has been suggested that targeting specific subtypes of leukemia may allow for more effective individualized treatment. However, the subtype of leukemia within an individual patient may change over time or as a result of treatment, and an individual patient may have multiple subclones.

[0005] Research has also been conducted into immunotherapeutic approaches to treat AML. Interleukin-3 receptor alpha chain (IL3RA, also known as CD123) was one of the first antigens targeted for the treatment of AML due to its overexpression on the vast majority of AML cells compared to normal bone marrow. Monoclonal antibodies and recombinant immunotoxins targeting CD123 have shown promise in preclinical evaluation. CD33 is another antigen of interest because it is expressed on more than 80% of AML malignant cells; however, it is also expressed on normal myeloid progenitor cell lines. Based on promising preclinical data, immunotherapies including CAR-T cells targeting these two surface molecules have been tested in early clinical trials in relapsed and refractory AML patients. However, preliminary clinical results were disappointing, as these approaches showed only short-term responses without long-term benefit against the disease and caused severe adverse effects due to the low specificity of the targets used, i.e., severe pancytopenia and severe bone marrow destruction due to expression of the targets also by hematopoietic stem and progenitor cells.

[0006] Therefore, there is a need for the development of safe and effective alternative therapies for the treatment of AML. Summary of the Invention [Means for solving the problem]

[0007] 1. Overview Using in silico analysis followed by experimental analysis and validation, applicants have identified six novel antigens (i.e., CD63, CD151, CD72, CD84, CD69 and CD109) specific to AML cells that have little to no expression within the hematopoietic stem cell compartment. These six tumor specific antigens (TSAs) were selected in part based on their stable, specific and high level expression in AML cells as measured by flow cytometry using AML cell lines (SHI-1, HL-60, KASUMI-1, MOLM-13, MV4-1 and AML).

[0008] Based on this discovery, the applicant provides methods for diagnosing and treating myeloid disorders and acute leukemia (e.g., AML) by targeting TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. Further provided herein are antigen binding proteins (ABPs), ABP-drug conjugates and chimeric antigen receptors (CARs) that can be used for these treatment methods.

[0009] Thus, the present disclosure provides a method of diagnosing myeloid disorder (MD) and acute leukemia (AL) in a subject, comprising detecting the presence or level of a tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0010] In some embodiments, the biological sample is a blood sample or a bone marrow sample. In some embodiments, the biological sample comprises blast cells. In some embodiments, the blast cells are selected from myeloid blast cells, lymphoid blast cells, or a combination of myeloid and lymphoid blast cells.

[0011] In some embodiments, the detecting step comprises contacting the biological sample with an antibody, wherein the antibody specifically binds to the tumor-specific antigen. In some embodiments, the detecting step comprises flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorophore or enzyme. In some embodiments, the target binding protein is labeled. In some embodiments, the target binding protein is labeled with a fluorophore or enzyme. In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample.

[0012] In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or next-generation sequencing.

[0013] In some embodiments, myeloid disorders (MD) and acute leukemia (AL) have onset in childhood or adult age. In some embodiments, the method further comprises determining the presence or absence of cancer cells in the subject based on detecting the presence or level of tumor-specific antigens in blood and / or bone marrow samples from the subject. In some embodiments, the method further comprises determining the presence or absence of cancer cells in the subject based on detecting the presence or level of tumor-specific antigens in biological samples from the subject.

[0014] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0015] In some embodiments, the therapeutic agent is an antigen binding protein (ABP), an ABP-drug conjugate, an immunoresponsive cell expressing a chimeric antigen receptor (CAR), or a bispecific T cell engager (BiTE), where the ABP, ABP-drug conjugate, CAR, or BiTE specifically binds to a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In some embodiments, the therapeutic agent is an ABP described in this disclosure, an ABP-drug conjugate described in this disclosure, an immunoresponsive cell expressing a chimeric antigen receptor (CAR) described in this disclosure, or a bispecific T cell engager (BiTE) described in this disclosure.

[0016] In another aspect, the disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), comprising the steps of: a) detecting the presence or level of a tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; b) administering to the subject a therapeutically effective amount of an immunoresponsive cell expressing an antigen binding protein (ABP), an ABP-drug conjugate, or a chimeric antigen receptor (CAR), wherein the ABP, ABP-drug conjugate, or CAR specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0017] In some embodiments, the biological sample is a blood sample, a bone marrow sample. In some embodiments, the biological sample comprises myeloid disorder (MD) and acute leukemia (AL) blast cells. In some embodiments, the blast cells are selected from myeloid blast cells, lymphoid blast cells, or a combination of myeloid blast cells and lymphoid blast cells.

[0018] In some embodiments, the presence or level of tumor-specific antigen is detected by contacting the biological sample with an antibody, which specifically binds to the tumor-specific antigen. In some embodiments, the presence or level of tumor-specific antigen is detected by flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence, or enzyme-linked immunosorbent assay (ELISA).

[0019] In some embodiments, the presence or level of tumor-specific antigen is detected by measuring the mRNA level of tumor-specific antigen in biological samples. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or next-generation sequencing.

[0020] In one aspect, the present disclosure provides an antigen binding protein (ABP) that specifically binds to a target protein selected from CD63, CD151, CD72, CD84, CD69, and CD109.

[0021] In some embodiments, the ABP specifically binds to human CD84.

[0022] In some embodiments, the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:90.

[0023] In some embodiments, the ABP is a. V having the sequence of SEQ ID NO:51 L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence SEQ ID NO: 61 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3; b. V having the sequence of SEQ ID NO:47L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3; or c. V having the sequence of SEQ ID NO:45 L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3 Includes.

[0024] In some embodiments, the ABP is d. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88; e. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88; or f. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:83. Includes.

[0025] In some embodiments, the ABP is a. V having the sequence of SEQ ID NO: 131 L CDR1, V having the sequence of SEQ ID NO: 132 L CDR2, V having the sequence of SEQ ID NO: 133 L CDR3, V having the sequence of SEQ ID NO: 251 H CDR1, V having the sequence of SEQ ID NO: 252 H CDR2, and V having the sequence of SEQ ID NO: 253 H CDR3; b. V having the sequence of SEQ ID NO: 134 L CDR1, V having the sequence of SEQ ID NO: 135 L CDR2, V having the sequence of SEQ ID NO: 136 L CDR3, V having the sequence of SEQ ID NO: 254 H CDR1, V having the sequence of SEQ ID NO: 255 H CDR2, and V having the sequence of SEQ ID NO: 256 H CDR3. c. V having the sequence of SEQ ID NO:110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2, and V having the sequence of SEQ ID NO: 232 H CDR3; d. V having the sequence of SEQ ID NO: 161 L CDR1, V having the sequence SEQ ID NO: 162 LCDR2, V having the sequence SEQ ID NO: 163 L CDR3, V having the sequence of SEQ ID NO: 281 H CDR1, V having the sequence of SEQ ID NO: 282 H CDR2, and V having the sequence of SEQ ID NO: 283 H CDR3; e. V having the sequence of SEQ ID NO: 164 L CDR1, V having the sequence of SEQ ID NO: 165 L CDR2, V having the sequence SEQ ID NO: 166 L CDR3, V having the sequence of SEQ ID NO: 284 H CDR1, V having the sequence of SEQ ID NO: 285 H CDR2, and V having the sequence of SEQ ID NO: 286 H CDR3; f. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 L CDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2, and V having the sequence of SEQ ID NO: 262 H CDR3; g. V having the sequence of SEQ ID NO: 191 L CDR1, V having the sequence of SEQ ID NO: 192 L CDR2, V having the sequence of SEQ ID NO: 193 L CDR3, V having the sequence of SEQ ID NO: 311 H CDR1, V having the sequence of SEQ ID NO: 312 H CDR2, and V having the sequence of SEQ ID NO: 313 H CDR3; h. V having the sequence of SEQ ID NO: 194 L CDR1, V having the sequence of SEQ ID NO: 195 L CDR2, V having the sequence of SEQ ID NO: 196 L CDR3, V having the sequence of SEQ ID NO: 314 H CDR1, V having the sequence of SEQ ID NO: 315 H CDR2, and V having the sequence of SEQ ID NO: 316 HCDR3; i. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290 H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2, and V having the sequence of SEQ ID NO: 292 H CDR3; j. V having the sequence of SEQ ID NO: 221 L CDR1, V having the sequence of SEQ ID NO: 222 L CDR2, V having the sequence of SEQ ID NO: 223 L CDR3, V having the sequence of SEQ ID NO: 341 H CDR1, V having the sequence of SEQ ID NO: 342 H CDR2, and V having the sequence of SEQ ID NO: 343 H CDR3; or V having the sequence of SEQ ID NO: 224 L CDR1, V having the sequence of SEQ ID NO: 225 L CDR2, V having the sequence of SEQ ID NO: 226 L CDR3, V having the sequence of SEQ ID NO: 344 H CDR1, V having the sequence of SEQ ID NO: 345 H CDR2, and V having the sequence of SEQ ID NO: 346 H CDR3; or k. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence SEQ ID NO: 201 L CDR2, V having the sequence SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320 H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2, and V having the sequence of SEQ ID NO: 322 H CDR3 Includes.

[0026] In some embodiments, the ABP specifically binds to human CD69.

[0027] In some embodiments, the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 89-96.

[0028] In some embodiments, the ABP is a. V having the sequence of SEQ ID NO:47 L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO:57 L CDR3, V having the sequence SEQ ID NO: 64 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2, and V having the sequence of SEQ ID NO: 71 H CDR3; b. V having the sequence of SEQ ID NO:50 L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO: 60 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO:69 H CDR2, and V having the sequence of SEQ ID NO: 73 H CDR3; c. V having the sequence of SEQ ID NO:45 L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 65 H CDR2, and V having the sequence of SEQ ID NO: 70 H CDR3; d. V having the sequence of SEQ ID NO:46 L CDR1, V having the sequence SEQ ID NO:53 L CDR2, V having the sequence of SEQ ID NO:56 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2, and V having the sequence of SEQ ID NO: 71H CDR3; e. V having the sequence of SEQ ID NO:48 L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO:58 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2, and V having the sequence of SEQ ID NO: 72 H CDR3; f. V having the sequence of SEQ ID NO:49 L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2, and V having the sequence of SEQ ID NO: 71 H CDR3; g. V having the sequence of SEQ ID NO:49 L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2, and V having the sequence of SEQ ID NO: 71 H CDR3; or h. V having the sequence of SEQ ID NO:45 L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 66 H CDR2, and V having the sequence of SEQ ID NO: 71 H CDR3 Includes.

[0029] In some embodiments, the ABP is a. V having the sequence of SEQ ID NO: 107 L CDR1, V having the sequence of SEQ ID NO: 108L CDR2, V having the sequence of SEQ ID NO: 109 L CDR3, V having the sequence of SEQ ID NO: 227 H CDR1, V having the sequence of SEQ ID NO: 228 H CDR2 and V having the sequence of SEQ ID NO: 229 H CDR3; b. V having the sequence of SEQ ID NO: 110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2, and V having the sequence of SEQ ID NO: 232 H CDR3; c. V having the sequence of SEQ ID NO: 113 L CDR1, V having the sequence of SEQ ID NO: 114 L CDR2, V having the sequence of SEQ ID NO: 115 L CDR3, V having the sequence of SEQ ID NO: 233 H CDR1, V having the sequence of SEQ ID NO: 234 H CDR2, and V having the sequence of SEQ ID NO: 235 H CDR3; d. V having the sequence of SEQ ID NO: 116 L CDR1, V having the sequence of SEQ ID NO: 117 L CDR2, V having the sequence of SEQ ID NO: 118 L CDR3, V having the sequence of SEQ ID NO: 236 H CDR1, V having the sequence of SEQ ID NO: 237 H CDR2, and V having the sequence of SEQ ID NO: 238 H CDR3; e. V having the sequence of SEQ ID NO: 119 L CDR1, V having the sequence of SEQ ID NO: 120 L CDR2, V having the sequence SEQ ID NO: 121 L CDR3, V having the sequence of SEQ ID NO: 239 H CDR1, V having the sequence of SEQ ID NO: 240 H CDR2, and V having the sequence of SEQ ID NO: 241H CDR3; f. V having the sequence of SEQ ID NO: 122 L CDR1, V having the sequence of SEQ ID NO: 123 L CDR2, V having the sequence of SEQ ID NO: 124 L CDR3, V having the sequence of SEQ ID NO: 242 H CDR1, V having the sequence of SEQ ID NO: 243 H CDR2, and V having the sequence of SEQ ID NO: 244 H CDR3; g. V having the sequence of SEQ ID NO: 125 L CDR1, V having the sequence of SEQ ID NO: 126 L CDR2, V having the sequence of SEQ ID NO: 127 L CDR3, V having the sequence of SEQ ID NO: 245 H CDR1, V having the sequence of SEQ ID NO: 246 H CDR2, and V H CDR3 having sequence of SEQ ID NO:247; h. V having the sequence of SEQ ID NO: 128 L CDR1, V having the sequence of SEQ ID NO: 129 L CDR2, V having the sequence of SEQ ID NO: 130 L CDR3, V having the sequence of SEQ ID NO: 248 H CDR1, V having the sequence of SEQ ID NO: 249 H CDR2, and V having the sequence of SEQ ID NO: 250 H CDR3; i. V having the sequence of SEQ ID NO: 137 L CDR1, V having the sequence of SEQ ID NO: 138 L CDR2, V having the sequence of SEQ ID NO: 139 L CDR3, V having the sequence of SEQ ID NO: 257 H CDR1, V having the sequence of SEQ ID NO: 258 H CDR2, and V having the sequence of SEQ ID NO: 259 H CDR3; j. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 LCDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2, and V having the sequence of SEQ ID NO: 262 H CDR3; k. V having the sequence of SEQ ID NO:143 L CDR1, V having the sequence of SEQ ID NO: 144 L CDR2, V having the sequence of SEQ ID NO: 145 L CDR3, V having the sequence of SEQ ID NO: 263 H CDR1, V having the sequence of SEQ ID NO: 264 H CDR2, and V having the sequence of SEQ ID NO: 265 H CDR3; l. V having the sequence of SEQ ID NO: 146 L CDR1, V having the sequence of SEQ ID NO: 147 L CDR2, V having the sequence of SEQ ID NO: 148 L CDR3, V having the sequence of SEQ ID NO: 266 H CDR1, V having the sequence of SEQ ID NO: 267 H CDR2, and V having the sequence of SEQ ID NO: 268 H CDR3; m. V having the sequence of SEQ ID NO: 149 L CDR1, V having the sequence of SEQ ID NO: 150 L CDR2, V having the sequence of SEQ ID NO: 151 L CDR3, V having the sequence of SEQ ID NO: 269 H CDR1, V having the sequence of SEQ ID NO: 270 H CDR2, and V having the sequence of SEQ ID NO: 271 H CDR3; n. V having the sequence of SEQ ID NO: 152 L CDR1, V having the sequence of SEQ ID NO: 153 L CDR2, V having the sequence of SEQ ID NO: 154 L CDR3, V having the sequence of SEQ ID NO: 272 H CDR1, V having the sequence of SEQ ID NO: 273 H CDR2, and V having the sequence of SEQ ID NO: 274 H CDR3; o. V having the sequence of SEQ ID NO: 155L CDR1, V having the sequence of SEQ ID NO: 156 L CDR2, V having the sequence of SEQ ID NO: 157 L CDR3, V having the sequence of SEQ ID NO: 275 H CDR1, V having the sequence of SEQ ID NO: 276 H CDR2, and V having the sequence of SEQ ID NO: 277 H CDR3; p. V having the sequence of SEQ ID NO: 158 L CDR1, V having the sequence of SEQ ID NO: 159 L CDR2, V having the sequence of SEQ ID NO: 160 L CDR3, V having the sequence of SEQ ID NO: 278 H CDR1, V having the sequence of SEQ ID NO: 279 H CDR2, and V having the sequence of SEQ ID NO: 280 H CDR3; q. V having the sequence of SEQ ID NO: 167 L CDR1, V having the sequence of SEQ ID NO: 168 L CDR2, V having the sequence of SEQ ID NO: 169 L CDR3, V having the sequence of SEQ ID NO: 287 H CDR1, V having the sequence of SEQ ID NO: 288 H CDR2, and V having the sequence of SEQ ID NO: 289 H CDR3; r. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290 H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2, and V having the sequence of SEQ ID NO: 292 H CDR3; s. V having the sequence of SEQ ID NO: 173 L CDR1, V having the sequence of SEQ ID NO: 174 L CDR2, V having the sequence of SEQ ID NO: 175 L CDR3, V having the sequence of SEQ ID NO: 293 H CDR1, V having the sequence of SEQ ID NO: 294H CDR2, and V having the sequence of SEQ ID NO: 295 H CDR3; t. V having the sequence of SEQ ID NO: 176 L CDR1, V having the sequence of SEQ ID NO: 177 L CDR2, V having the sequence of SEQ ID NO: 178 L CDR3, V having the sequence of SEQ ID NO: 296 H CDR1, V having the sequence of SEQ ID NO: 297 H CDR2, and V having the sequence of SEQ ID NO: 298 H CDR3; u. V having the sequence of SEQ ID NO: 179 L CDR1, V having the sequence of SEQ ID NO: 180 L CDR2, V having the sequence of SEQ ID NO: 181 L CDR3, V having the sequence of SEQ ID NO: 299 H CDR1, V having the sequence of SEQ ID NO: 300 H CDR2, and V having the sequence of SEQ ID NO: 301 H CDR3; v. V having the sequence of SEQ ID NO: 182 L CDR1, V having the sequence of SEQ ID NO: 183 L CDR2, V having the sequence SEQ ID NO: 184 L CDR3, V having the sequence of SEQ ID NO: 302 H CDR1, V having the sequence of SEQ ID NO: 303 H CDR2, and V having the sequence of SEQ ID NO: 304 H CDR3; w. V having the sequence of SEQ ID NO: 185 L CDR1, V having the sequence of SEQ ID NO: 186 L CDR2, V having the sequence of SEQ ID NO: 187 L CDR3, V having the sequence of SEQ ID NO: 305 H CDR1, V having the sequence of SEQ ID NO: 306 H CDR2, and V having the sequence of SEQ ID NO: 307 H CDR3; x. V having the sequence of SEQ ID NO: 188 L CDR1, V having the sequence of SEQ ID NO: 189 LCDR2, V having the sequence of SEQ ID NO: 190 L CDR3, V having the sequence of SEQ ID NO: 308 H CDR1, V having the sequence of SEQ ID NO: 309 H CDR2, and V having the sequence of SEQ ID NO: 310 H CDR3; y. V having the sequence of SEQ ID NO: 197 L CDR1, V having the sequence of SEQ ID NO: 198 L CDR2, V having the sequence of SEQ ID NO: 199 L CDR3, V having the sequence of SEQ ID NO: 317 H CDR1, V having the sequence of SEQ ID NO: 318 H CDR2, and V having the sequence of SEQ ID NO: 319 H CDR3; z. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence SEQ ID NO: 201 L CDR2, V having the sequence SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320 H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2, and V having the sequence of SEQ ID NO: 322 H CDR3; aa. V having the sequence of SEQ ID NO: 203 L CDR1, V having the sequence of SEQ ID NO: 204 L CDR2, V having the sequence of SEQ ID NO: 205 L CDR3, V having the sequence of SEQ ID NO: 323 H CDR1, V having the sequence of SEQ ID NO: 324 H CDR2, and V having the sequence of SEQ ID NO: 325 H CDR3; bb. V having the sequence of SEQ ID NO: 206 L CDR1, V having the sequence of SEQ ID NO: 207 L CDR2, V having the sequence of SEQ ID NO: 208 L CDR3, V having the sequence of SEQ ID NO: 326 H CDR1, V having the sequence of SEQ ID NO: 327 H CDR2, and V having the sequence of SEQ ID NO: 328 HCDR3; cc. V having the sequence of SEQ ID NO: 209 L CDR1, V having the sequence of SEQ ID NO: 210 L CDR2, V having the sequence of SEQ ID NO: 211 L CDR3, V having the sequence of SEQ ID NO: 329 H CDR1, V having the sequence of SEQ ID NO: 330 H CDR2, and V having the sequence of SEQ ID NO: 331 H CDR3; dd. V having the sequence of SEQ ID NO: 212 L CDR1, V having the sequence of SEQ ID NO: 213 L CDR2, V having the sequence of SEQ ID NO: 214 L CDR3, V having the sequence of SEQ ID NO: 332 H CDR1, V having the sequence of SEQ ID NO: 333 H CDR2, and V having the sequence of SEQ ID NO: 334 H CDR3; ee. V having the sequence of SEQ ID NO: 215 L CDR1, V having the sequence of SEQ ID NO: 216 L CDR2, V having the sequence of SEQ ID NO: 217 L CDR3, V having the sequence of SEQ ID NO: 335 H CDR1, V having the sequence of SEQ ID NO: 336 H CDR2, and V having the sequence of SEQ ID NO: 337 H CDR3; or ff. V having the sequence of SEQ ID NO: 218 L CDR1, V having the sequence of SEQ ID NO: 219 L CDR2, V having the sequence of SEQ ID NO: 220 L CDR3, V having the sequence of SEQ ID NO: 338 H CDR1, V having the sequence of SEQ ID NO: 339 H CDR2, and V having the sequence of SEQ ID NO: 340 H CDR3 Includes.

[0030] In some embodiments, the ABP is a. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; b. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:79 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:87; c. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83; d. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:82; e. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; f. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86; g. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; h. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; or i. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83. Includes.

[0031] In some embodiments, the ABP specifically binds to human CD69 and CD84.

[0032] In some embodiments, the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:36.

[0033] In some embodiments, the ABP has the sequence of SEQ ID NO:45. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0034] In some embodiments, the ABP comprises a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:83.

[0035] In some embodiments, the ABP comprises an amino acid sequence selected from SEQ ID NOs:89-98.

[0036] In some embodiments, the ABP is a Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 , single chain antibody molecules, dual variable domain antibodies, single variable domain antibodies, linear antibodies or V domain antibodies.

[0037] In some embodiments, the ABP is a Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 , a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody or a V domain antibody.

[0038] In some embodiments, the ABP is a monoclonal antibody.

[0039] In some embodiments, the ABP is selected from an IgG, an IgM, an IgA, an IgD, and an IgE antibody.

[0040] In some embodiments, the ABP comprises a heavy chain constant region of class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4.

[0041] In some embodiments, the ABP is conjugated to a drug.

[0042] In some embodiments, the ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, and comprises a human Fc.

[0043] In some embodiments, the ABP is a human, humanized or chimeric ABP.

[0044] In some embodiments, the ABP is monoclonal.

[0045] In some embodiments, the ABP is bispecific or multispecific.

[0046] In some embodiments, the ABP comprises the heavy chain constant region of an IgG.

[0047] In some embodiments, the ABP is defucosylated.

[0048] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D It binds to the target protein.

[0049] In one aspect, the present disclosure provides an isolated polynucleotide or set of polynucleotides encoding the ABP described in this disclosure.In another aspect, the present disclosure provides a vector or set of vectors comprising the isolated polynucleotide described in this disclosure.In another aspect, the present disclosure provides a host cell comprising the isolated polynucleotide or vector described in this disclosure.

[0050] In one aspect, the present disclosure provides a method of producing an isolated antigen binding protein (ABP), the method comprising expressing the ABP in a host cell described in the present disclosure and isolating the ABP.

[0051] In one aspect, the disclosure provides an antigen binding protein (ABP) capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, and comprising a human Fc.

[0052] In some embodiments, the ABP is a human, humanized or chimeric ABP. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific. In some embodiments, the ABP comprises an IgG heavy chain constant region. In some embodiments, the ABP comprises an IgG1 heavy chain constant region. In some embodiments, the ABP is defucosylated.

[0053] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D It binds to the target protein.

[0054] In another aspect, the present disclosure provides a pharmaceutical composition comprising an ABP provided herein and a pharma- ceutically acceptable excipient.

[0055] In yet another aspect, the present disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), comprising administering a therapeutically effective amount of an ABP or pharmaceutical composition provided herein. In some embodiments, the myeloid disorder (MD) and acute leukemia (AL) are of pediatric or adult onset.

[0056] In some embodiments, the ABP or pharmaceutical composition is administered in combination with an additional agent. In some embodiments, the additional agent is a chemotherapeutic agent or a biological agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacytidine, decitabine, and CPX-351 (Vyxeos®). In some embodiments, the additional agent is a hedgehog pathway inhibitor. In some embodiments, the hedgehog pathway inhibitor is a sonic hedgehog pathway inhibitor. In some embodiments, the sonic hedgehog pathway inhibitor is selected from vismodegib, sonidigib, and arsenic trioxide (ATO). In some embodiments, the hedgehog pathway inhibitor is glasdegib (Daurismo™). In some embodiments, the additional agent is an FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from the group consisting of midostaurin (Rydapt®), gilteritinib (Xospata®), sorafenib, lestaurtinib, quizartinib, and crenolanib. In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib (Tibsovo®) or enasidenib (Idhifa®). In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax (Venclexta®). In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg™) or vadastuximab talirine (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor.In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0057] In some embodiments, the ABP or pharmaceutical composition is not administered in combination with an immunotherapy. In some embodiments, the ABP or pharmaceutical composition is not administered by combination therapy. In some embodiments, the ABP or pharmaceutical composition is not administered by immunotherapy. In some embodiments, the method comprises administering a therapeutically effective amount of an immunoresponsive cell comprising a CAR, and the administering step is not followed or combined with autologous or allogeneic hematopoietic stem cell therapy to support hematopoiesis.

[0058] In yet another aspect, the disclosure provides an antigen binding protein (ABP)-drug conjugate comprising an antigen binding protein (ABP), a cytotoxic agent linked to the ABP, and optionally a linker linking the cytotoxic agent to the ABP, wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0059] In some embodiments, the ABP is a human, humanized or chimeric ABP. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific. In some embodiments, the ABP is a Fab, Fab", F(ab") 2 , Fv, scFv, (scFv) 2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, a V domain antibody, or a bispecific tandem bivalent scFv, or a bispecific T cell engager (BiTE). In some embodiments, the ABP comprises an Fc, optionally a human Fc. In some embodiments, the ABP comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the ABP comprises a heavy chain constant region of a class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG1.

[0060] In certain embodiments, the ABP is a BiTE.In certain embodiments, the BiTE comprises an antigen binding domain and a T cell activation domain.In certain embodiments, the antigen binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0061] In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0062] In some embodiments, the antigen binding domain binds to an epitope on the CD63, CD151, CD72, CD84, CD69, or CD109 target antigen. In some embodiments, the antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the antigen binding domain comprises the VDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. H and V L In some embodiments, the antigen binding domain comprises a CD63, CD151, CD72, CD84, CD69, or CD109 single chain variable fragment (scFv).

[0063] In some embodiments, the T cell activation domain comprises the intracellular domain of CD3ζ. In certain embodiments, the T cell activation domain binds to CD3.

[0064] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D It binds to the target protein.

[0065] In some embodiments, the cytotoxic agent comprises an antiangiogenic agent, a proapoptotic agent, an antimitotic agent, an antikinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioisotope. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0066] One aspect of the present disclosure provides a pharmaceutical composition comprising an ABP-drug conjugate described in the present disclosure and a pharma- ceutically acceptable excipient.

[0067] In one aspect, the present disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), comprising administering a therapeutically effective amount of an ABP-drug conjugate or pharmaceutical composition described in the present disclosure.

[0068] In some embodiments, the myeloid disorder (MD) and acute leukemia (AL) are of pediatric or adult onset. In some embodiments, the ABP-drug conjugate or pharmaceutical composition is administered in combination with an additional agent. In some embodiments, the additional agent is a chemotherapeutic agent or a biological agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacytidine, decitabine, and CPX-351 (Vyxeos®). In some embodiments, the additional agent is a hedgehog pathway inhibitor. In some embodiments, the hedgehog pathway inhibitor is a sonic hedgehog pathway inhibitor. In some embodiments, the sonic hedgehog pathway inhibitor is selected from vismodegib, sonidegib, and arsenic trioxide (ATO). In some embodiments, the hedgehog pathway inhibitor is glasdegib (Daurismo™). In some embodiments, the additional agent is an FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from the group consisting of midostaurin (Rydapt®), gilteritinib (Xospata®), sorafenib, lestaurtinib, quizartinib, and crenolanib. In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib (Tibsovo®) or enasidenib (Idhifa®). In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax (Venclexta®). In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg™) or vadastuximab butarilin (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor.In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0069] In yet another aspect, the disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, a signaling domain and optionally a costimulatory domain, wherein the extracellular antigen binding domain specifically binds to a target protein or antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0070] In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein.

[0071] In some embodiments, the extracellular antigen binding domain comprises an ABP described in this disclosure.

[0072] In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ.

[0073] In some embodiments, the CAR further comprises a costimulatory domain, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, a CD27 costimulatory domain, an OX40 costimulatory domain, or an ICOS costimulatory domain.

[0074] In some embodiments, the costimulatory domain is a 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 105.

[0075] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0076] In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:100.

[0077] In some embodiments, the CAR further comprises a hinge region.

[0078] In some embodiments, the hinge region is a hinge region derived from a CD28 polypeptide.

[0079] In some embodiments, the hinge region comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:99.

[0080] In some embodiments, the signaling domain is a CD3 zeta signaling domain.

[0081] In some embodiments, the CD3 zeta signaling domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:101.

[0082] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-44.

[0083] In some embodiments, the extracellular antigen-binding domain specifically binds human CD84.

[0084] In some embodiments, the extracellular antigen-binding domain specifically binds to human CD69.

[0085] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid selected from SEQ ID NOs: 35-42.

[0086] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acids of SEQ ID NO: 43, 44 or 36.

[0087] In one aspect, the disclosure provides a polynucleotide encoding a CAR described in this disclosure.

[0088] In some embodiments, the CAR comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to the nucleotide sequence of SEQ ID NOs: 24-34.

[0089] In one aspect, the disclosure provides a vector comprising a CAR polynucleotide of the disclosure.

[0090] In one aspect, the present disclosure provides an immunoresponsive cell that expresses a CAR described in this disclosure.

[0091] In one aspect, the present disclosure provides an immunoresponsive cell comprising a CAR polynucleotide described in this disclosure or a vector described in this disclosure.

[0092] In some embodiments, the immunoresponsive cell is an αβ T cell, a γδ T cell, or a natural killer (NK) cell.

[0093] In some embodiments, the αβ T cells are CD4 +T cells or CD8 + T cells.

[0094] In one aspect, the present disclosure provides a method of preparing an immunoresponsive cell described in this disclosure, comprising transfecting or transducing a CAR polynucleotide or vector described in this disclosure into an immune cell.

[0095] In some embodiments, the method includes expanding the immune cells for at least 48 hours.

[0096] In some embodiments, immune cells are transduced at a multiplicity of infection ranging from 1-100.

[0097] In some embodiments, the method further comprises, after the transducing step, washing the vector and expanding the transduced immune cells for at least 2 days. In some embodiments, the method further comprises, after the transducing step, washing the vector and expanding the transduced immune cells for a period ranging from 2 to 30 days.

[0098] In one aspect, the present disclosure provides a method of treating a subject, the method comprising administering a therapeutically effective amount of an ABP described in this disclosure, an ABP-drug conjugate described in this disclosure, a pharmaceutical composition described in this disclosure, or an immunoresponsive cell described in this disclosure.

[0099] In some embodiments, the subject has a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, the acute leukemia is acute lymphoblastic leukemia (ALL).

[0100] In some embodiments, the myeloid disorders (MD) and acute leukemia (AL) are of childhood or adult onset.

[0101] In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein. In some embodiments, the signaling domain comprises an intracellular domain of CD3zeta. In some embodiments, the CAR further comprises a costimulatory domain, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, a CD27 costimulatory domain, an OX40 costimulatory domain, or an ICOS costimulatory domain.

[0102] In one aspect, the present disclosure provides a polynucleotide encoding a CAR. In another aspect, the present disclosure provides a vector comprising the polynucleotide. In yet another aspect, the present disclosure provides an immunoresponsive cell expressing a CAR described in the present disclosure and an immunoresponsive cell comprising a polynucleotide.

[0103] In some embodiments, the immunoresponsive cells are αβ T cells, γδ T cells, or natural killer (NK) cells. In some embodiments, αβ T cells are CD4+ T cells, CD3 + T cells or CD8+ T cells.

[0104] In one aspect, the disclosure provides a method of preparing an immunoresponsive cell, the method comprising transfecting or transducing a polynucleotide or vector into the immunoresponsive cell.

[0105] In another aspect, the disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), comprising administering a therapeutically effective amount of an immunoresponsive cell. In some embodiments, the myeloid disorder (MD) and acute leukemia (AL) are of pediatric or adult onset.

[0106] In some embodiments, the immunoresponsive cells are administered in combination with an additional agent. In some embodiments, the additional agent is administered before or after the step of administering a therapeutically effective amount of the immunoresponsive cells. In some embodiments, the additional agent is administered concurrently with the step of administering a therapeutically effective amount of the immunoresponsive cells. In some embodiments, the additional agent is administered before the step of administering a therapeutically effective amount of the immunoresponsive cells. In some embodiments, the additional agent is administered after the step of administering a therapeutically effective amount of the immunoresponsive cells.

[0107] In some embodiments, the immunoresponsive cells are administered in combination with an additional agent. In some embodiments, the additional agent is a chemotherapeutic agent or a biological agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, decitabine, and CPX-351 (Vyxeos®). In some embodiments, the additional agent is a hedgehog pathway inhibitor. In some embodiments, the hedgehog pathway inhibitor is a sonic hedgehog pathway inhibitor. In some embodiments, the sonic hedgehog pathway inhibitor is selected from vismodegib, sonidegib, and arsenic trioxide (ATO). In some embodiments, the hedgehog pathway inhibitor is glasdegib (Daurismo™). In some embodiments, the additional agent is an FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from the group consisting of midostaurin (Rydapt®), gilteritinib (Xospata®), sorafenib, lestaurtinib, quizartinib, and crenolanib. In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib (Tibsovo®) or enasidenib (Idhifa®). In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax (Venclexta®). In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg™) or vadastuximab butarilin (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor.In some embodiments, the additional agent is an immune checkpoint inhibitor, hi some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0108] In some embodiments, the method of treatment further comprises treating the subject with a chemotherapeutic agent or hematopoietic stem cells prior to administering the ABP, pharmaceutical composition or immunoresponsive cells. In some embodiments, the method of treatment further comprises treating the subject with a chemotherapeutic agent or hematopoietic stem cells after administering the ABP, pharmaceutical composition or immunoresponsive cells. In some embodiments, the administering step is not followed by or combined with immunoglobulin therapy. In some embodiments, the immunoglobulin therapy is intravenous immunoglobulin (IVIG) treatment.

[0109] In some embodiments, the subject has refractory disease. In some embodiments, the subject has relapse. In some embodiments, the subject is an adult AML patient. In some embodiments, the subject is a pediatric AML patient.

[0110] In some embodiments, prior to administering a therapeutically effective amount of an ABP, pharmaceutical composition, or immunoresponsive cell to the subject, the subject has been administered a chemotherapeutic agent or has undergone hematopoietic stem cell therapy.

[0111] In some embodiments, the subject is non-responsive to chemotherapy or hematopoietic stem cell therapy.

[0112] In some embodiments, the subject has a myeloblastic (M0) type of AML. In some embodiments, the subject has a myeloblastic (M1) type of AML. In some embodiments, the subject has a myeloblastic (M2) type of AML. In some embodiments, the subject has a promyeloytic (M3) type of AML. In some embodiments, the subject has a myelomonocytic (M4) type of AML. In some embodiments, the subject has a monocytic (M5) type of AML. In some embodiments, the subject has an erythroleukemic (M6) type of AML. In some embodiments, the subject has a megakaryocytic (M7) type of AML.

[0113] In some embodiments, the method of treatment comprises administering an effective amount of immunoresponsive cells comprising a CAR that targets CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the effective amount is a dose ranging from 100,000 cells / kg to 25 million cells / kg. In some embodiments, the effective amount is a dose ranging from 100,000 cells / kg to 25 million cells / kg. In some embodiments, the effective amount is a dose ranging from 100,000 cells / kg to 15 million cells / kg.

[0114] In one aspect, the disclosure provides a bispecific T cell engager (BiTE) comprising an antigen binding domain and a T cell activation domain, wherein the antigen binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0115] In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein.

[0116] In some embodiments, the T cell activation domain comprises the intracellular domain of CD3ζ.

[0117] In some embodiments, the T cell activation domain comprises a single chain variable fragment (scFV) of an antibody that specifically binds CD3.

[0118] In another aspect, the disclosure provides a polynucleotide encoding a BiTE.In another aspect, the disclosure provides a vector comprising a polynucleotide encoding a BiTE.

[0119] In another aspect, the disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), comprising administering a therapeutically effective amount of a BiTE.

[0120] In some embodiments, the myeloid disorders (MD) and acute leukemia (AL) are of childhood or adult onset.

[0121] In some embodiments, the BiTE or vector is administered in combination with an additional agent.

[0122] In some embodiments, the additional agent is a chemotherapeutic agent or a biological agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacytidine, decitabine, and CPX-351 (Vyxeos®). In some embodiments, the additional agent is a hedgehog pathway inhibitor. In some embodiments, the hedgehog pathway inhibitor is a sonic hedgehog pathway inhibitor. In some embodiments, the sonic hedgehog pathway inhibitor is selected from vismodegib, sonidegib, and arsenic trioxide (ATO). In some embodiments, the hedgehog pathway inhibitor is glasdegib (Daurismo™). In some embodiments, the additional agent is an FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from the group consisting of midostaurin (Rydapt®), gilteritinib (Xospata®), sorafenib, lestaurtinib, quizartinib, and crenolanib. In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib (Tibsovo®) or enasidenib (Idhifa®). In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax (Venclexta®). In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg™) or vadastuximab butarilin (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor.In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0123] 2. Brief description of some drawings These and other features, aspects, and advantages of the present disclosure will become better understood with reference to the following description and the accompanying drawings. [Brief description of the drawings]

[0124] [Figure 1] Figure 1 is a flow chart showing the process of identifying novel AML TSAs as described in Example 1. The screening flow chart shows the original two lists of genes selected from AML gene expression data at diagnosis, along with prioritization based on specific inclusion and exclusion criteria that defined 26 / 76 genes that were carried forward to flow cytometry expression analysis in AML cell lines (SHI-1, HL-60 MOLM-13, MV4;11 and Kasumi-1) and healthy control hematopoietic cells (PBMCs sorted for CD3+, CD19+ and CD33+ subpopulations and CD34+ cells isolated from umbilical cord blood). The best candidate TSA localization on the AML cell surface was confirmed by immunofluorescence.

[0125] [Figure 2-1]FIG. 2A shows data from a flow cytometry analysis performed with two different antibodies against CD69 (light grey histogram) versus isotype (dark grey histogram) on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11). Cytometry analysis data for isotype is provided as a control (dark grey histogram). Isotype controls are antibodies that maintain similar properties to the primary antibody but lack specific target binding. FIG. 2B shows images of fluorescent immunohistochemistry (white dots) of an AML cell line (SHI-1) with two antibodies against CD69, together with membrane staining (Membrite: colocalization of CD69 with plasma membrane-specific dyes). FIG. 2C shows flow cytometry data from an analysis performed with CD69 antibodies on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations derived from PBMCs and CD34-positive cells derived from umbilical cord blood. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0126] [Figure 3-1] Figure 3A shows data from a flow cytometry analysis performed with two different antibodies against CD63 on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) (light grey histograms). Cytometry analysis data for isotypes are provided as a control (dark grey histograms). Figure 3B shows images of fluorescent immunohistochemistry (white dots) of an AML cell line (HL-60) with two antibodies against CD63, together with membrane staining (Membrite: colocalization of CD63 with a plasma membrane specific dye). Figure 3C shows data from a flow cytometry analysis performed with CD63 antibody on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations from PBMC and CD34 positive cells from umbilical cord blood. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above.

[0127] [Figure 4-1] Figure 4A shows data from a flow cytometry analysis performed with two different antibodies against CD151 on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) (light grey histograms). Cytometry analysis data for isotypes are provided as a control (dark grey histograms). Figure 4B shows images of fluorescent immunohistochemistry (white dots) of an AML cell line (HL-60) with two antibodies against CD151, together with membrane staining (Membrite: colocalization of CD151 with a plasma membrane specific dye). Figure 4C shows data from a flow cytometry analysis performed with CD151 antibodies on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations from PBMC and CD34 positive cells from umbilical cord blood. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0128] [Figure 5-1] Figure 5A shows data from flow cytometry analysis performed with two different antibodies against CD84 on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) (light grey histograms). Cytometry analysis data for isotypes are provided as a control (dark grey histograms). Figure 5B shows images of fluorescent immunohistochemistry (white dots) of an AML cell line (HL-60) with two antibodies against CD84, together with membrane staining (Membrite: colocalization of CD84 with plasma membrane specific dyes). Figure 5C shows data from flow cytometry analysis performed with CD84 antibody on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations from PBMC and CD34 positive cells from umbilical cord blood. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0129] [Figure 6-1] Figure 6A shows data from flow cytometry analysis performed with two different antibodies against CD109 on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) (light grey histograms). Cytometry analysis data for isotypes are provided as a control (dark grey histograms). Figure 6B shows images of fluorescent immunohistochemistry (white dots) of AML cell lines (Kasumi-1 or HL-60) with two antibodies against CD109, together with membrane staining (Membrite: colocalization of CD109 with plasma membrane-specific dyes). Figure 6C shows data from flow cytometry analysis performed with CD109 antibody on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations derived from PBMCs and CD34-positive cells derived from umbilical cord blood. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0130] [Figure 7-1] Figure 7A shows data from flow cytometry analysis performed with two different antibodies against CD72 on several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) (light grey histograms). Cytometry analysis data for isotypes are provided as a control (dark grey histograms). Figure 7B shows images of fluorescent immunohistochemistry (white dots) of an AML cell line (SHI-1) with two antibodies against CD72, together with membrane staining (Membrite: colocalization of CD72 with a plasma membrane specific dye). Figure 7C shows data from flow cytometry analysis performed with CD72 antibody on healthy hematopoietic cells, i.e. healthy CD3, CD19 and CD33 subpopulations from PBMC and CD34 positive cells from umbilical cord blood. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0131] [Figure 8-1] FIG. 8 shows flow cytometry results on human primary dermal fibroblasts to test the specificity of the TSA. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 8-6] Same as above.

[0132] [Figure 9-1] Figure 9 shows flow cytometry results for different cancer cell lines to test the specificity of TSA. TSA expression levels were assessed in cell lines from different cancer types by flow cytometry and are shown as histograms (light grey) versus isotype control (grey). [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above.

[0133] [Figure 10-1] FIG. 10 shows flow cytometry results for AML cell lines using different commercially available antibodies. [Figure 10-2] Same as above.

[0134] [Figure 11]Figure 11 shows TSA mRNA expression in AML samples collected at diagnosis and in remission after treatment. TSA mRNA expression was assessed by quantitative PCR in 14 paired pediatric AML bone marrow samples collected at diagnosis (RQ=1) and in remission after treatment (dots indicate mRNA expression values ​​at remission versus at diagnosis). Patients are stratified by genetic risk of AML (SR=standard risk; HR=high risk). Despite heterogeneity in TSA mRNA expression, a significant (* indicates p<0.05) reduction in CD69, CD63 and CD84 mRNA expression after treatment was detected (RQ<1).

[0135] [Figure 12-1] Figures 12A-12B show TSA expression by flow cytometry in the clinic. Figure 12A shows the lack of CD69 and CD84 expression in the CD34+CD38- subpopulation. Figure 12B shows TSA expression in AML samples at diagnosis, showing the highest expression of CD69 and CD84 in AML blasts. [Figure 12-2] Same as above.

[0136] [Figure 13] Pipeline for selection and construction of novel AML CARs against CD69 and CD84. Ten ScFv sequences were selected from phage display and prioritized based on specificity for cloning into the CAR cassette in a third generation lentiviral transfer plasmid for expression.

[0137] [Figure 14]CAR-T cell manufacturing workflow. Schematic representation of the lentiviral transduction process and CAR-T cell manufacturing. The workflow includes the isolation of donor T cells on day 1, efficient activation by TransAct, and gene transfer of CAR-LV constructs on day 3. From day 4, CAR-T cells (generated with ScFv sequences B8 or F12 for CD84 and G1 or H3 for CD69) underwent expansion growth in TexMACS medium supplemented with IL-7 and IL-15 for 14 days. On day 17, CAR-T cells were immunophenotyped and grown in co-culture with HL-60 or SHI-1 (CD84+CD69+) AML cell lines.

[0138] [Figure 15] Transduction efficiency was determined on day 17 by measuring vector copy number (VCN, number of integrated viral copies per cell). VCN was determined by digital droplet PCR (ddPCR) analysis using primers within the lentiviral backbone for all CAR-T cell constructs for all experiments performed. Each symbol represents a different CAR construct (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]).

[0139] [Figure 16-1] Flow cytometry cell surface expression of CD69 and CD84 on the indicated AML cell lines, as well as isotype controls (dark grey peaks). Representative histograms of three independent experiments. [Figure 16-2] Same as above.

[0140] [Figure 17]Representative expansion fold kinetics of T cells during CAR-T cell manufacturing (day 0-14). Mock-transduced (empty CAR) and T cells were monitored and cell numbers were calculated by flow cytometry. Data are shown as mean ± SEM (n = 2-7, P > 0.05). Each symbol indicates a different CAR construct (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock-transduced [empty CAR]).

[0141] [Figure 18] Frequency of viable T lymphocytes (Figure 18A) and percentage of CD4+ and CD8+ cells within lymphocytes at the end of production (day 14) measured by flow cytometry (Figure 18B). Bars and symbols indicate different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]). Data are shown as mean ± SEM of 2-5 different experiments.

[0142] [Figure 19] Representative flow cytometry plots (Figure 19A) and frequencies of T cell subsets (Figure 19B) from day 1 to the end of CAR-T cell manufacturing (day 14). Immunophenotypes were determined by flow cytometry: naive (Tn) and stem cell memory (Tscm), CD197(CCR7)+CD45RO-; central memory (Tcm), CD197(CCR7)+CD45RO+; terminally differentiated (Tef), CD197(CCR7)-CD45RO-; effector memory (Tem) and transitional memory (Ttm), CD197(CCR7)-CD45RO+. Bars indicate different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]).

[0143] [Figure 20-1] The immunophenotype of CAR-T cells to assess their activation (Figure 20A) and exhaustion (Figure 20B) by expression of the indicated markers was determined by flow cytometry on days 1 and 17. Data show the mean ± SEM of 1 to 4 different experiments. Bars indicate the different CAR-T cell products (those generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and those mock transduced [empty CAR]). [Figure 20-2] Same as above.

[0144] [Figure 21] T cells engineered to express CD84 (F12, B8) and CD69 (G1, H3) CARs recognize and kill target AML cell lines. Target HL-60 and SHI-1 (CD84+CD69+) AML cell lines were grown in TexMACS medium (--) or in co-culture with effector CAR-T cells at an E:T ratio of 1:1 for 48 hours. Target AML cell lines stained with 7AAD and Annexin-V (monitored by CD33+) were analyzed by flow cytometry. SHI-1 and HL-60 cell lines had a higher percentage of cell death when co-cultured with CAR-T cells than when co-cultured with mock-transduced cells (empty CAR) (*P<0.05, **P<0.01 vs. empty CAR). Bars indicate different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and empty CAR). Data are shown as mean ± SEM of 1 to 4 different experiments.

[0145] [Figure 22]Each bar represents the percentage of killing (% lytic efficacy) achieved by a different CAR-T product against the target AML cell line. Bars represent different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]). Data are shown as mean ± SEM of 1-3 different experiments (*P<0.05 vs. empty CAR). CAR-T cell lytic efficacy is calculated as follows:

number

[0146] [Diagram 23] Percentage of CAR-T cell lysis measured by bioluminescence (BLI) in luciferase (LUC)-transduced target AML cell lines. Each bar shows the percentage of killing achieved by different CAR-T products against AML-LUC cell lines, normalized with respect to the BLI value of the relevant AML cell line cultured alone. The bars represent different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock-transduced [empty CAR]). Data are shown as the mean ± SEM of two different experiments (***P<0.001; ****P<0.0001 vs. empty CAR).

[0147] [Figure 24] Absolute number of live CAR-T cells cultured alone (-) or with target AML cell lines at an effector:target (E:T) ratio of 1:1 for 48 hours, quantified by flow cytometry. Data show effector cell persistence and lysis of target AML cells. Dotted line shows CAR-T cells at day 17 before co-culture. Each symbol represents a different CAR-T cell product (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]) and cell line.

[0148] [Diagram 25] Representative images (Figure 25A) and absolute numbers of (Figure 25B) colony forming units (CFU) generated from CD34+ cells cultured alone (---) or with CAR-T cells at an E:T ratio of 1:1 for 6 hours and then plated in MethoCult for 14 days. Bars and symbols indicate different CAR-T cell products (generated with ScFv sequences B8 or F12 for CD84, G1 or H3 for CD69, and mock transduced [empty CAR]) and human CD34+ cells. Data are shown as mean ± SEM of 2-3 different experiments (ns not significant at p>0.05).

[0149] [Figure 26-1] Luciferase bioluminescence signals observed in leukemic mouse xenografts are provided in FIG. 26A. The effect of CD69(H3) CAR-T cells on the growth of CD69+ AML cell lines in leukemic mouse xenografts is shown. FIG. 26B provides the in vivo lytic potency of the corresponding AML cell lines by CAR-T cells as indicated by luciferase signal reduction (shown as total flux) (*P<0.05 vs. mock-transduced cells [empty CAR]). FIG. 26C shows the in vivo lytic potency of CD69 (H3 and G1) and CD84 (B8 and F12) CAR-T cells against CD69+CD84+ AML cell lines (*P<0.05 vs. empty CAR). NSG mice were injected with 0.5×106 SHI-1-LUC cells and 2 days later with 1.5×106 CAR-T (1:3 ratio. N=7 animals / group). Data are presented as mean±SEM. AML engraftment and spread was monitored weekly by luciferase bioluminescence. [Figure 26-2] Same as above.

[0150] [Figure 27-1]Figures 27A-27D provide the in vivo efficacy of B8 and F12 ScFv chains directed against CD84, and H3 ScFv directed against CD69. The in vivo lytic potency of the corresponding CAR-T cells against SHI-1 targeted AML cell lines is shown by luciferase signal reduction (shown as total flux). (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 Mann-Whitney test). NSG mice were injected with 0.5x106 SHI-1 luciferase positive cells and 2 days later with 1.5x106 CAR-T (1:3 ratio, n=3-7 animals / group). Survival of NSG mice injected with SHI-1 (CD84+CD69+) AML cell line, and (FIG. 27C) F12 / B8 or empty CAR-T cells (no scFv), and (FIG. 27D) H3 or empty CAR-T cells. Data are shown as mean±SEM. AML engraftment and spread was monitored weekly by luciferase bioluminescence; *p<0.05 Mantel-Cox test. [Figure 27-2] Same as above.

[0151] [Figure 28] Figures 28A-28B provide the in vivo specificity of B8 and F12 ScFv chains directed against CD84. Effect of anti-CD84 (B8) CAR-T cells against (Figure 28A) CD84negK562 and (Figure 28B) CD84negU937 AML cell lines. In vivo lytic potency of CAR-T cells against non-targeted AML cell lines is indicated by luciferase signal reduction (shown as total flux). NSG mice were injected with 0.5x106 AML-LUC+ cells and 2 days later with 1.5x106 CAR-T (1:3 ratio. n=6-10 animals / group). Data are shown as mean ± SEM. AML engraftment and spread was monitored weekly by luciferase bioluminescence.

[0152] [Figure 29]Figure 29 provides expression of novel TSAs in primary AML cells derived from patient-derived xenografts (AML-PDX). Median fluorescence intensity (MFI) of CD84, CD69 and CD72 cell surface expression on AML cells harvested from PDX models generated from pediatric AML samples at the time of AML de novo diagnosis by flow cytometry (isotype control, left peak - dark grey; AML, right peak - light grey). Representative histograms of two independent experiments.

[0153] [Diagram 30] Figure 30 provides the in vitro lytic efficacy of anti-CD84 CAR-T cells against primary AML cells. The percentage of AML cells killed by CAR T cells generated to express anti-CD84 (except B8 for PDX#5) ScFv when co-cultured with ex vivo target primary cells collected from AML-PDX models (1:1 effector:target ratio, 48 hours). Ex vivo target AML primary cells stained with 7AAD and Annexin-V (monitored by CD33 expression) were analyzed by flow cytometry (lytic efficacy was normalized to that induced by empty CAR T cells).

[0154] [Diagram 31] Figures 31A-31B provide in vitro lytic potency of anti-CD69 A1, F1, C2, H2 ScFv(s). Lytic potency induced by anti-CD69 A1, F1, C2, H2 ScFv(s) when co-cultured with target (Figure 31A) SHI-1 and (Figure 31B) HL60 AML CD84+CD69+ cell lines for 48 hours and measured by flow cytometry by 7AAD and Annexin-V expression. Each bar shows the percentage of killing achieved by different CAR-T cell products against cell lines normalized to cell lines when CAR-T cells were cultured alone (---) and against empty CAR T cells.

[0155] [Diagram 32]Figures 32A-32B show the in vitro cytokine production capacity of anti-CD84 B8, F12 ScFv(s) CAR T cells. Percentage of (Figure 32A) IFNγ and (Figure 32B) TNFα positive expressing cells after 48 hours of co-culture of empty or anti-CD84 B8 or F12 CAR T cells with target SHI-1 and HL60 (CD84+ / CD69+) AML cell lines at an E:T ratio of 1:1, as measured by flow cytometry. Each bar represents the mean ± SEM of 1-4 independent experiments. (*p<0.05, **p<0.005, ***p<0.0005 Mann-Whitney T-test).

[0156] [Diagram 33] Figures 33A-33B show in vitro off-target cytotoxicity results of B8 and F12 ScFv chains directed against CD84 ScFv when co-cultured with CD34+ HSCs. Representative experiments of (Figure 33A) absolute number of total colony forming units (CFU) and (Figure 34B) colony morphology generated by CD34+ HSCs of CAR-T cells cultured alone (---) or with CAR-T cells at a 1:1 E:T ratio for 6 hours and then plated in MethoCult medium for 12 days to differentiate multipotent granulocyte, erythrocyte, macrophage and megakaryocyte colonies, granulocyte-macrophage colonies, granulocyte colonies and macrophage colonies (CFU-GEMM, CFU-GM, CFU-G and CFU-M, respectively) (data shown as mean ± SEM, n=1-2, not significant at p>0.05).

[0157] [Diagram 34]Figures 34A-34B provide the in vivo specificity of B8 and F12 ScFv chains directed against CD84. (Figure 34A) Effect of anti-CD84 (B8) CAR-T cells on SHI-1-CD84ko AML cell line. In vivo lytic potency of CAR-T cells is indicated by luciferase signal reduction (shown as total light flux). NSG mice were injected with 0.5x106 AML-LUC+SHI-1ko cells and treated with 1.5x106 CAR-T on days 2 and 8 (E:T 1:3, n=4 animals / group). Data are shown as mean ± SEM. Engraftment and spread of SHI-1ko was monitored weekly by luciferase bioluminescence. (Figure 34B) Survival of NSG mice injected with SHI-1-CD84ko AML cell line and F12 / B8 or empty CAR-T cells. (Data are shown as mean ± SEM, not significant at p>0.05).

[0158] [Diagram 35] Figures 35A-35B provide the in vitro killing capacity of B8 CD84 and F12 CD84 CAR-T cells and A1 CD69 C2 CD68 CAR-T cells. Percentage of AML cells killed by CAR T cells generated to express (Figure 35A) anti-CD84 (B8 and F12) or (Figure 35B) anti-CD69 (A1 and C2) ScFv chains when co-cultured with target primary ex vivo cells collected from AML-PDX models (E:T ratio 1:1, 48 hours). Histograms showing cell death of AML primary cells (monitored by CD33 expression and stained with 7AAD and Annexin-V) by flow cytometry. Lytic potency was normalized to the lytic potency induced by empty CAR T cells. Each histogram shows the mean ± SEM of each replicate (n = 1-2 replicates per group).

[0159] [Diagram 36]Figures 36A-36B provide in vitro cytokine production capacity of anti-CD84 B8, F12 ScFv(s). (Figure 36A) IFNγ and (Figure 36B) TNFα production of CAR T cells after 48 hours of co-culture at 1:1 E:T ratio with target CD84+ AML primary cells derived from AML-PDX models as measured by flow cytometry. Data are normalized to cytokine production of CAR T cells in medium alone (---). Representative experiment (not significant at p>0.05).

[0160] [Figure 37-1] Figures 37A-37C provide in vivo efficacy of B8 ScFv chains directed to AML-PDX expressing CD84. (Figure 37A) NSG mice were injected with 1.0x106 AML-luciferase (LUC) expressing cells and AML engraftment and spread was monitored weekly by LUC bioluminescence (shown as total light flux). On day +28 from AML injection, LUC analysis yielded a reliable signal of AML engraftment and mice were tail injected with 5x106 CAR-T cells (5:1 E:T ratio) or 10x106 CAR-T cells (10:1 E:T ratio, n=5-10 animals / group). (Figure 37B-37C) Distribution of frequency of CAR T cells (% lymphocyte CD3+) and PDX-AML cells in peripheral blood (PB), bone marrow (BM) and spleen (SPL) of mice injected with AML-PDX and CAR-T cells on (Figure 37B) day 7 or (Figure 37C) days 12 and 16 after CAR-T cell inoculation. (nd = not detected, data shown as mean ± SEM of 1-2 mice per group). [Figure 37-2] Same as above.

[0161] [Figure 38]Figures 38A-38B provide the in vivo efficacy of B8 ScFv chains directed to AML-PDX cells expressing CD84. (Figure 38A) NSG mice were tail-injected with 1.0x106 AML-luciferase (LUC) expressing cells and 2 days later treated with 5x106 CAR-T cells (E:T ratio of 5:1) or 10x106 CAR-T cells (E:T ratio of 10:1, n=5-10 animals / group). (Figure 38B) Distribution of lymphocyte and PDX-AML cell frequency in peripheral blood (PB), bone marrow (BM), spleen (SPL) and lung (LNG) of AML-PDX and CAR-T cell injected mice 12 days after CAR-T inoculation. (Data shown as mean ± SEM of 1-2 mice per group).

[0162] [Figure 39-1] Figures 39A-39H provide in vivo off-target effects of B8 ScFv chains on hematopoietic precursors. (Figure 39A) NSG mice were engrafted with 106 human CD34+ hematopoietic stem cells (HSCs) after sublethal irradiation and checked weekly for human leukocyte engraftment by flow cytometry. At week +8 post-transplant, mice were tail vein injected with 3x106 empty or anti-CD84 B8 CAR T cells and monitored daily for variations in peripheral blood leukocyte composition from day +1 to day +8 post-injection. (Figure 39B) Representative flow cytometry strategy to monitor leukocyte engraftment in the peripheral blood of mice transplanted with hCD34+HSCs. (Figures 39C-39H) Variation in frequency of cells of human hematopoietic precursors identified by flow cytometry in the peripheral blood of mice (n=1) after empty CAR or B8 CAR T cell infusion. [Figure 39-2] Same as above. [Figure 39-3] Same as above. [Figure 39-4] Same as above.

[0163] [Figure 40-1]Figures 40A-40K show predictions of the binding interface between CAR binding factors (ScFv) and CD69 (Figures 40A-40H) or CD84 (Figures 40I-40K). Using ChimeraX software (v.1.5), molecular models of ScFv are presented in complex with the extracellular domain of CD69 or CD84 (sequence from uniprot); contact residues in the sequence are displayed and highlighted. Figures 40A-40H show predicted structures of the CD69 extracellular domain bound to scFv A1, C1, F1, G1, C2, F2, H2 or H3 (top), as well as the amino acid sequence of the scFv with the binding domains highlighted (bottom). The results show that scFv A1, C1, F1, G1, C2, F2, H2 and H3 bind similarly to the extracellular domain of CD69. Amino acid chain docking is similar between scFvs A1, C1, F1, G1, C2, F2, H2 and H3, and extends to the entire extracellular domain of the ScFv(s) as well as the entire extracellular domain of CD69. Figures 40I-40K show the predicted structure of the CD84 extracellular domain bound to scFvs B8 and F12 (top), as well as the amino acid sequence of the scFv with the binding domains highlighted (bottom). These results show that ScFvs B8 and F12 bind to the extracellular domain of CD84 in a similar manner. Amino acid chain docking is similar between scFvs B8 and F12, and extends to the entire extracellular domain of the ScFv(s) as well as the entire extracellular domain of CD84. [Figure 40-2] Same as above. [Figure 40-3] Same as above. [Figure 40-4] Same as above. [Figure 40-5] Same as above. [Figure 40-6] Same as above. [Figure 40-7] Same as above. [Figure 40-8] Same as above. [Figure 40-9] Same as above. [Figure 40-10] Same as above. [Figure 40-11] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0164] 3. Detailed Description 3.1.Definition Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. In general, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are well known and commonly used in the art. The methods and techniques of this disclosure are generally carried out according to conventional methods well known in the art, unless otherwise indicated, and as described in various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The terminology used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, and the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0165] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0166] Unless further modified by the name of the non-human species, the terms "CD63," "CD63 protein," and "CD63 antigen" are used interchangeably herein to refer to human CD63, or any variants (e.g., splice and allelic variants), isoforms, or species orthologs of human CD63 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD63 gene (NCBI Accession Number: NG_008347, Gene ID 967).

[0167] Unless further modified by the name of a non-human species, the terms "CD151," "CD151 protein," and "CD151 antigen" are used interchangeably herein to refer to human CD151, or any variants (e.g., splice and allelic variants), isoforms, or species orthologs of human CD151 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD151 gene (NCBI Accession Number: NG_007478.1, Gene ID 977).

[0168] Unless further modified by the name of the non-human species, the terms "CD72," "CD72 protein," and "CD72 antigen" are used interchangeably herein to refer to human CD72, or any variants (e.g., splice and allelic variants), isoforms, or species orthologs of human CD72 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD72 gene (NCBI Accession Number: NC_000009.12, Gene ID 971).

[0169] Unless further modified by the name of the non-human species, the terms "CD84," "CD84 protein," and "CD84 antigen" are used interchangeably herein to refer to human CD84, or any variants (e.g., splice and allelic variants), isoforms, or species orthologs of human CD84 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD84 gene (NCBI Accession Number: NC_000001.11, Gene ID 8832).

[0170] Unless further modified by the name of the non-human species, the terms "CD69," "CD69 protein," and "CD69 antigen" are used interchangeably herein to refer to human CD69, or any variant (e.g., splice variants and allelic variants), isoforms, or species orthologs of human CD69 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD69 gene (NCBI Accession Number: NC_000012.12, Gene ID 969).

[0171] Unless further modified by the name of the non-human species, the terms "CD109," "CD109 protein," and "CD109 antigen" are used interchangeably herein to refer to human CD69, or any variant (e.g., splice variants and allelic variants), isoforms, or species orthologs of human CD109 that are naturally expressed by cells, including neoplastic cells, or that are expressed by cells transfected with the CD109 gene (NCBI Accession Number: NC_000006.12, Gene ID 135228).

[0172] The term "antigen binding protein" (ABP) refers to a protein that comprises one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. For example, "CD63", "anti-CD63 ABP" or "CD63-specific ABP" is an ABP provided herein that specifically binds to the antigen CD63. In some embodiments, the ABP binds to the extracellular domain of CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, the CD63, CD151, CD72, CD84, CD69, or CD109 ABPs provided herein bind to an epitope of CD151, CD72, CD84, CD69, or CD109, respectively, that is conserved between or among CD151, CD72, CD84, CD69, or CD109 proteins from different species.

[0173] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is the V H -V L The antigen-binding domain is formed by a dimer. Antibodies are one type of ABP.

[0174] The term "antigen-binding domain" refers to a portion of an ABP capable of specifically binding to an antigen or epitope.

[0175] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain, including an Fc region.

[0176] The term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain that interacts with the Fc receptor and certain proteins of the complement system in naturally occurring antibodies. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or a modified Fc region as described elsewhere in this disclosure.

[0177] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab') 2 These include fragments, Fab' fragments, scFv (sFv) fragments and scFv-Fc fragments.

[0178] An "Fv" fragment comprises a non-covalently linked dimer of one heavy- and one light-chain variable domain.

[0179] A "Fab" fragment contains the heavy and light chain variable domains as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H 1) Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0180] "F(ab') 2The F(ab')" fragment contains two Fab' fragments joined near the hinge region by disulfide bonds. 2 Fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0181] A "single-chain Fv" or "sFv" or "scFv" antibody fragment has one V in a single polypeptide chain. H Domain and one V L Includes domain. V H and V L are generally linked by a peptide linker. See Pluckthun A. (1994). In some embodiments, the linker is (GGGGS)n (SEQ ID NO:1). In some embodiments, n=1, 2, 3, 4, 5 or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.

[0182] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain can be linked to the C-terminus of the scFv. H -V L or V L -V H ) depending on V H or V L Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0183] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen, with no other variable domains present.

[0184] A "monospecific ABP" is an ABP that contains a binding site that specifically binds to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule that is bivalent but recognizes the same epitope in each antigen-binding domain. The binding specificity can be present in any suitable valency.

[0185] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, yeast clones, bacterial clones or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0186] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0187] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequences derived from non-human antibodies. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, camelid or non-human primate antibody, with the desired specificity, affinity or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further improve antibody function.

[0188] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or derived from a non-human source using a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0189] An "isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been separated and / or recovered from a component of its natural environment. The components of the natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials. In some embodiments, the isolated ABP is purified to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by use of a spinning cup sequenator. In some embodiments, the isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions with detection by Coomassie blue or silver staining. An isolated ABP includes an ABP in situ in a recombinant cell, since at least one component of the ABP's natural environment is absent. In some embodiments, the isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95% or 99% by weight. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95% or 99% by volume. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution containing at least 85%, 90%, 95%, 98%, 99% to 100% by volume of ABP or nucleic acid. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution containing at least 85%, 90%, 95%, 98%, 99% to 100% by volume of ABP or nucleic acid.

[0190] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an ABP and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D The affinity can be expressed by the kinetic constant (Eq. (2)). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. The affinity can be measured by common methods known in the art, including those described herein. The affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®), or by monoclonal competitive ELISA testing.

[0191] With respect to the binding of ABPs to target molecules, the terms "bind", "specific binding", "specifically binds to", "specific for", "selectively binds" and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics a recognized epitope on the target molecule. In that case, specific binding is indicated when binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the CD63, CD151, CD72, CD84, CD69 or CD109 ABP for a non-target molecule is less than about 50% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 40% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 30% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 20% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of a CD63, CD151, CD72, CD84, CD69 or CD109 ABP for a non-target molecule is less than about 10% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively.In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for a non-target molecule is less than about 1% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for a non-target molecule is less than about 0.1% of its affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively.

[0192] The term “kd” (seconds -1 ) as used herein refers to the dissociation rate constant of a particular ABP-antigen interaction. This value is also called the k value.

[0193] The term "ka" (M -1 × seconds -1 ) as used herein refers to the association rate constant of a particular ABP-antigen interaction. This value is also referred to as the k value.

[0194] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. K D =kd / ka.

[0195] The term “K A " " -1 ) as used herein refers to the association equilibrium constant of a particular ABP-antigen interaction. A =ka / kd.

[0196] An "affinity matured" ABP is an ABP that has one or more changes (e.g., in one or more CDRs or FRs) that result in an improvement in the affinity of the ABP to its antigen, compared to a parent ABP that does not have the changes. In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity to the target antigen. Affinity matured ABPs can be produced using various methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, incorporated by reference in its entirety) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described, for example, by Barbas et al. (Proc. Nat. Acad. Sci. USA, 1994, 91:3809-3813); Schier et al., Gene, 1995, 169:147-155; Yelton et al., J. Immunol., 1995, 155:1994-2004; Jackson et al., J. Immunol., 1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226:889-896, each of which is incorporated by reference in its entirety.

[0197] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecule(s).

[0198] "Effector function" refers to the biological activity mediated by the Fc region of an antibody, and these activities may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

[0199] As used herein with respect to two or more ABPs, the term "competes with" or "cross-competes with" indicates that two or more ABPs compete for binding to an antigen (e.g., CD63, CD151, CD72, CD84, CD69 or CD109). In one exemplary assay, CD63, CD151, CD72, CD84, CD69 or CD109 is coated on a surface and contacted with a first CD63, CD151, CD72, CD84, CD69 or CD109 ABP, respectively, followed by the addition of a second CD63, CD151, CD72, CD84, CD69 or CD109 ABP. In another exemplary assay, a first CD63, CD151, CD72, CD84, CD69 or CD109 ABP is coated on a surface and contacted with CD63, CD151, CD72, CD84, CD69 or CD109, and then a second CD63, CD151, CD72, CD84, CD69 or CD109 ABP is added. In either assay, if the presence of the first CD63, CD151, CD72, CD84, CD69 or CD109 ABP reduces the binding of the second CD63, CD151, CD72, CD84, CD69 or CD109 ABP, these ABPs compete with each other. The term "competes with" also includes combinations of ABPs where one ABP reduces the binding of another ABP, but no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit each other's binding, regardless of the order in which they are added. In some embodiments, one ABP reduces the binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. Those skilled in the art can select the concentration of the antibody used in the competition assay based on the affinity of the ABP to CD63, CD151, CD72, CD84, CD69 or CD109 and the valency of the ABP. The assays described in this definition are exemplary, and those skilled in the art can utilize any suitable assay to determine whether antibodies compete with each other.Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, each of which is incorporated by reference in its entirety.

[0200] The term "epitope" refers to the portion of an antigen that specifically binds to ABP. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter may not be lost. Epitopes may include amino acid residues that are directly involved in binding, and other amino acid residues that are not directly involved in binding. The epitope that ABP binds to may be determined using known techniques for epitope determination, such as testing ABP binding to CD63, CD151, CD72, CD84, CD69 or CD109 variants with different point mutations, or chimeric CD63, CD151, CD72, CD84, CD69 or CD109 variants.

[0201] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0202] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease, and remission, or improved prognosis.

[0203] As used herein, the term "subject" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with the immunoresponsive cell comprising the ABP, ABP-drug conjugate or CAR provided herein. In some embodiments, the disease or condition is cancer.

[0204] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0205] The term "myeloid" as used herein includes all cells belonging to the granulocyte (i.e., neutrophils, eosinophils, basophils), monocyte / macrophage, erythrocyte, megakaryocyte and mast cell lineages. Myeloid malignancies are clonal disorders of hematopoietic stem or progenitor cells. These malignancies can be present in bone marrow and peripheral blood. They can result from genetic and epigenetic alterations that disrupt vital processes, such as self-renewal, proliferation and impaired differentiation. 3.2. Other Interpretive Conventions

[0206] Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0207] Unless otherwise indicated, a reference to a compound having one or more stereocenters contemplates each and every stereoisomer thereof, and all combinations of stereoisomers. 3.3. Compositions Targeting Tumor-Specific Antigens

[0208] One aspect of the present disclosure relates to a protein that targets a tumor-specific antigen selected from CD63, CD151, CD72, CD84, CD69 and CD109.

[0209] In some embodiments, the tumor-specific antigen is CD63. In some embodiments, CD63 is a protein encoded by the CD63 gene (12q13.2) (NCBI accession number: NG_008347, gene ID 967).

[0210] In some embodiments, the tumor-specific antigen is CD151. In some embodiments, CD151 is a protein encoded by the CD151 gene (11p15.5) (NCBI accession number: NG_007478.1, gene ID 977). Multiple alternatively spliced ​​transcript variants that code for the same protein have been described for this gene. Any of the splice variants can be used in various embodiments.

[0211] In some embodiments, the tumor-specific antigen is CD72. In some embodiments, CD72 is a protein encoded by the CD72 gene (9p13.3) (NCBI accession number: NC_000009.12, gene ID 971).

[0212] In some embodiments, the tumor-specific antigen is CD84. In some embodiments, CD84 is a protein encoded by the CD84 gene (1q23.3) (NCBI accession number: NC_000001.11, gene ID 8832).

[0213] In some embodiments, the tumor-specific antigen is CD69. In some embodiments, CD69 is a protein encoded by the CD69 gene (12p13.31) (NCBI accession number: NC_000012.12, gene ID 969).

[0214] In some embodiments, the tumor-specific antigen is CD109. In some embodiments, CD109 is a protein encoded by the CD109 gene (6q13) (NCBI accession number: NC_000006.12, gene ID 135228). 3.3.1. Antigen Binding Proteins (ABPs)

[0215] In one aspect, the disclosure provides an antigen binding protein (ABP) that specifically binds to CD63, CD151, CD72, CD84, CD69, or CD109.

[0216] In certain embodiments, the ABP specifically binds to CD63. In certain embodiments, the ABP specifically binds to CD151. In certain embodiments, the ABP specifically binds to CD72. In certain embodiments, the ABP specifically binds to CD84. In certain embodiments, the ABP specifically binds to CD69. In certain embodiments, the ABP specifically binds to CD109. In some embodiments, the ABP is an isolated antigen binding protein (ABP) that specifically binds to human CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, the ABP specifically binds to CD84 or CD69.

[0217] In some embodiments, the ABP comprises a human Fc.

[0218] In some embodiments, the ABP is a human, humanized or chimeric ABP.

[0219] In certain embodiments, the ABP is monoclonal.

[0220] In some embodiments, the ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) when administered. In some embodiments, the ABP binds to cell surface CD63, CD151, CD72, CD84, CD69, or CD109, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, natural killer (NK) cells result in ADCC by binding the Fc domain of the ABP to CD16 on the NK cell surface.

[0221] In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP comprises an immunoglobulin constant region. The antibody fragment can also be any synthetic or genetically engineered protein. For example, the antibody fragment includes an isolated fragment of the light chain variable region, an "Fv" fragment of the heavy and light chain variable regions, and a recombinant single chain polypeptide molecule (scFv protein) in which the light and heavy chain variable regions are connected by a peptide linker.

[0222] Another form of antibody fragment is a peptide that contains one or more complementarity determining regions (CDRs) of an antibody. CDRs (also called "minimal recognition units" or "hypervariable regions") can be incorporated into a molecule either covalently or non-covalently to make the molecule an antigen-binding protein. CDRs can be obtained by constructing a polynucleotide that codes for the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies, in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995)).

[0223] Thus, in one embodiment, the antibody fragment comprises at least one CDR as described herein. The binding agent may comprise at least two, three, four, five or six CDRs as described herein. The antibody fragment may further comprise at least one variable region domain of an antibody as described herein. The variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence responsible for binding to human CD63, CD151, CD72, CD84, CD69 or CD109, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as specifically described herein, which are adjacent to or in frame with one or more framework sequences. In general terms, the variable (V) region domain is a CDR sequence that is associated with an immunoglobulin heavy (V H ) and / or light (V L ) chain variable domains. Thus, for example, the V region domains can be monomeric and can be V regions capable of independently binding to a target antigen with an affinity in the range of 1 nM to 1 pM. H or V L Alternatively, the V region domain may be dimeric, H V H , V H V L or V L V L A V region dimer may comprise at least one V region that may be non-covalently associated. H A chain and at least one V L chain (hereinafter F V Optionally, these chains are combined into a single chain Fv (scF V The variable domains can be covalently coupled, for example, directly via a disulfide bond between the two variable domains or via a linker, such as a peptide linker, to form a 3'-amino acid linker (AAAA).

[0224] The variable region domain can be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include those created from a specific antibody variable region, for example, by insertion, deletion or change in or to the amino acid sequence of the specific antibody. Particular examples include engineered variable region domains that contain at least one CDR and, optionally, one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0225] The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, a V H The V domain may be linked to an immunoglobulin CH1 domain or a fragment thereof. L The domains can be linked to a CK domain or a fragment thereof. In this way, for example, an antibody can be produced comprising an associated VIII domain in which the antigen binding domain is covalently linked at their C-termini to the CH1 and CK domains, respectively. H and V L The CH1 domain may be an Fab fragment containing the CH1 domain, e.g., the CH1 domain may be extended with additional amino acids to provide a hinge region or a portion of the hinge region domain found in a Fab' fragment, or to provide additional domains, e.g., antibody CH2 and CH3 domains.

[0226] As described herein, an antibody comprises at least one of these CDRs. For example, one or more CDRs can be incorporated into known antibody framework regions (IgG1, IgG2, etc.) or conjugated to a suitable vehicle to enhance its half-life. Suitable vehicles include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, etc. These and other suitable vehicles are known in the art. Such conjugated CDR peptides can be monomeric, dimeric, tetrameric or other forms. In one embodiment, one or more water-soluble polymers are attached to one or more specific positions of the binder, for example, at the amino terminus.

[0227] ABPs of the present disclosure that specifically bind to CD84 or CD69 are listed in Table 10 (V L and V H CDRs) and Table 11 (light and heavy chain variable regions). [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

[0228] In some embodiments, the ABP comprises a light chain variable domain (V) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 45-51 in Table 10. L ) CDR1.

[0229] In some embodiments, the ABP comprises a light chain variable domain (V) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 52-54 in Table 10. L ) CDR2.

[0230] In some embodiments, the ABP comprises a light chain variable domain (V) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 55-61 in Table 10. L ) CDR3.

[0231] In some embodiments, the ABP comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 45-51 in Table 10 (a). L CDR1; (b) a V having an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 52 to 54 L CDR2; and (c) a V that includes an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 55 to 61. L Includes CDR3.

[0232] In some embodiments, the ABP is (a) V L V comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR1 amino acid sequences. L CDR1; (b) V in Table 10 LV comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR2 amino acid sequences. L CDR2; and (c) V of Table 10 L V comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR3 amino acid sequences. L Includes CDR3.

[0233] In some embodiments, the ABP is (a) V H A light chain variable domain (V) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR1 amino acid sequences. H In some embodiments, the ABP comprises (b) CDR1 of Table 10. H V comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR2 amino acid sequences. H In some embodiments, the ABP comprises CDR2. H V comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the CDR3 amino acid sequences. H Includes CDR3.

[0234] In some embodiments, the ABP comprises (a) a light chain variable domain (V) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 62-64. HIn some embodiments, the ABP comprises (b) a V CDR1 comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 65-69. H In some embodiments, the ABP comprises a V CDR2 comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 70-73. H Includes CDR3.

[0235] In some embodiments, the ABP comprises (a) an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SNTASWN (SEQ ID NO: 62); SNSASWN (SEQ ID NO: 63); and STTASWN (SEQ ID NO: 64). H CDR1; (b) a V that contains an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 65 to 69 H CDR2; and (c) a V that includes an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 70 to 73. H Includes CDR3.

[0236] In some embodiments, the ABP comprises CDR sequences identical to an antibody selected from A1, C1, F1, G1, C2, F2, H2, H3, F12, and B8.

[0237] In some embodiments, the ABP has the sequence of SEQ ID NO:45. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 LCDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 65 H CDR2 and V having the sequence of SEQ ID NO: 70 H Includes CDR3.

[0238] In some embodiments, the ABP has the sequence of SEQ ID NO:45. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0239] In some embodiments, the ABP has the sequence of SEQ ID NO:46. L CDR1, V having the sequence SEQ ID NO:53 L CDR2, V having the sequence of SEQ ID NO:56 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0240] In some embodiments, the ABP has the sequence of SEQ ID NO:47. L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO:57 L CDR3, V having the sequence SEQ ID NO: 64 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0241] In some embodiments, the ABP has the sequence of SEQ ID NO:48. L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO:58L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H Includes CDR3.

[0242] In some embodiments, the ABP has the sequence of SEQ ID NO:49. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0243] In some embodiments, the ABP has the sequence of SEQ ID NO:49. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0244] In some embodiments, the ABP has the sequence of SEQ ID NO:50. L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence of SEQ ID NO: 60 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO:69 H CDR2 and V having the sequence of SEQ ID NO: 73 H Includes CDR3.

[0245] In some embodiments, the ABP has the sequence of SEQ ID NO:47. L CDR1, V having the sequence SEQ ID NO:54 LCDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H Includes CDR3.

[0246] In some embodiments, the ABP has the sequence of SEQ ID NO:51. L CDR1, V having the sequence SEQ ID NO:54 L CDR2, V having the sequence SEQ ID NO: 61 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H Includes CDR3.

[0247] In some embodiments, the ABP has the sequence of SEQ ID NO:45. L CDR1, V having the sequence SEQ ID NO:52 L CDR2, V having the sequence of SEQ ID NO:55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H Includes CDR3.

[0248] In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP is a monoclonal antibody. In some embodiments, the ABP is selected from a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the ABP is a single chain variable fragment (scFv). In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0249] In some embodiments, the ABP comprises a variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 74-88 of Table 11. [Table 11-1] [Table 11-2]

[0250] In some embodiments, the ABP comprises a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 82-88 of Table 11.

[0251] In some embodiments, the ABP comprises a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 74-81 of Table 11.

[0252] In some embodiments, the ABP comprises a heavy chain variable domain and a light chain variable domain of an antibody selected from A1, C1, F1, G1, C2, F2, H2, H3, F12, and B8.

[0253] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:82 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0254] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:83 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0255] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:75.

[0256] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:85 and a light chain variable domain having the sequence of SEQ ID NO:76.

[0257] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:86 and a light chain variable domain having the sequence of SEQ ID NO:77.

[0258] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:77.

[0259] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:78.

[0260] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:87 and a light chain variable domain having the sequence of SEQ ID NO:79.

[0261] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:88 and a light chain variable domain having the sequence of SEQ ID NO:80.

[0262] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:88 and a light chain variable domain having the sequence of SEQ ID NO:81.

[0263] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:83 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0264] In some embodiments, the ABP comprises an scFv. In some embodiments, the ABP is an scFv. In some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 89-98 in Table 12. [Table 12-1] [Table 12-2] [Table 12-3]

[0265] In certain embodiments, the ABP is a Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 , a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, a V domain antibody, a bispecific tandem bivalent scFv or a bispecific T cell engager (BiTE).

[0266] In certain embodiments, the ABP comprises an Fc, optionally a human Fc.

[0267] In some embodiments, the ABP comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE and IgM. In some embodiments, the ABP comprises a heavy chain constant region of a class IgG and a subclass selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG. In certain embodiments, the ABP comprises a heavy chain constant region of IgG1.

[0268] In some embodiments, the ABP is bispecific or multispecific, hi some embodiments, the ABP is defucosylated.

[0269] Also provided are kits that include one or more of the pharmaceutical compositions comprising the ABP and instructions for use of the pharmaceutical compositions. Pharmaceutical compositions that include the ABPs described herein are also provided.

[0270] Also provided are isolated polynucleotides encoding the ABPs provided herein, or portions thereof. Also provided are vectors comprising such polynucleotides. Also provided are recombinant host cells comprising such polynucleotides and recombinant host cells comprising such vectors.

[0271] Also provided is a method for producing ABP using the polynucleotide, vector or host cell provided herein.In some aspects, the present disclosure provides a method for producing ABP that specifically binds to human CD63, CD151, CD72, CD84, CD69 or CD109, comprising expressing ABP in host cell and isolating ABP. 3.3.2.ABP-Drug Conjugates

[0272] Another aspect of the present disclosure provides antigen binding protein (ABP)-drug conjugates.

[0273] In some embodiments, the ABP-drug conjugate comprises an antigen binding protein (ABP), a cytotoxic agent linked to the ABP, and optionally a linker linking the cytotoxic agent to the ABP, wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0274] In some embodiments, the ABP-drug conjugate comprises one or more of the antigen binding proteins (ABPs) described in 3.3.1.

[0275] In certain embodiments, the ABP specifically binds to CD63. In certain embodiments, the ABP specifically binds to CD151. In certain embodiments, the ABP specifically binds to CD72. In certain embodiments, the ABP specifically binds to CD84. In certain embodiments, the ABP specifically binds to CD69. In certain embodiments, the ABP specifically binds to CD109.

[0276] In some embodiments, the ABP is a human, humanized or chimeric ABP. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific.

[0277] In some embodiments, the ABP is a Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 , single chain antibody molecules, dual variable domain antibodies, single variable domain antibodies, linear antibodies or V domain antibodies.

[0278] In some embodiments, the ABP comprises an Fc, optionally a human Fc.

[0279] In some embodiments, the ABP comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE and IgM. In some embodiments, the ABP comprises a heavy chain constant region of class IgG and a subclass selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG1. In some embodiments, the ABP comprises a variable heavy chain region. In some embodiments, the ABP comprises a variable light chain region. In some embodiments, the ABP comprises a variable heavy chain region and a variable light chain region. In some embodiments, the ABP is an antibody binding fragment. Antibodies and antigen binding fragments are described in 3.4.1 "Antigen Binding Proteins (ABPs)".

[0280] In some embodiments, the cytotoxic agent comprises an anti-angiogenic agent, a pro-apoptotic agent, a mitotic inhibitor, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioisotope.

[0281] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0282] In some embodiments, the backbone of the linker is 100 atoms or less in length, for example, 50 atoms or less in length, or 20 atoms or less in length. In other embodiments, the backbone of the linker is 100 atoms or more in length. The linker or linkage can be a covalent bond connecting two groups, or a chain between 1 and 100 atoms in length, for example, between 1 and 50 atoms in length or between 1 and 20 atoms in length, for example, about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length, and the linker can be linear, branched, cyclic, or single atom. In certain embodiments, 1, 2, 3, 4, or 5 or more carbon atoms of the linker backbone can be optionally replaced with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms can be saturated or unsaturated, and usually no more than 1, 2, or 3 unsaturated bonds are present in the linker backbone. The linker may include one or more substituents, for example, alkyl, aryl or alkenyl groups. The linker may include, but is not limited to, oligo(ethylene glycol); ether, thioether, tertiary amine, alkyl that may be linear or branched, for example, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone may include a cyclic group, for example, an aryl, heterocyclic or cycloalkyl group, and two or more atoms of the cyclic group, for example, 2, 3 or 4 atoms, are included in the backbone. The linker may be peptidic or non-peptidic.

[0283] Non-limiting examples of antibody-drug conjugate linkers and chemistries can be found in U.S. Patent Application Publication Nos. 20200277306 and 20190328900 A1; and U.S. Patent No. 7,750,116 B1, which are hereby incorporated by reference in their entireties. Additional non-limiting examples of ADC chemistries are described in Olivier et al., (2017) "Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer" ISBN: 978-1-119-06068-0), which are hereby incorporated by reference in their entireties.

[0284] Aspects of the present disclosure include the polynucleotide that codes for ABP-drug conjugate.In some embodiments, the polynucleotide further comprises a sequence that is homologous to the target genome region for site-specific integration.In some embodiments, the polynucleotide is designed for gene editing using endonucleases, such as CRISPR-Cas system, zinc finger nuclease, transcription activator-like effector nuclease (TALEN) and meganuclease.

[0285] In some embodiments, the polynucleotide is in a viral or non-viral vector. The vector can be used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0286] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide, or a pharmaceutical composition thereof, hi some embodiments, the non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0287] Non-limiting examples of non-viral delivery methods that can be used in the methods of the invention to deliver polynucleotides include, but are not limited to, physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical vectors, inorganic particles, calcium phosphate particles, magnetic particles, polymer-based vectors, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-co-glycoside) (PLGA), dendrimers, or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06) and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are hereby incorporated by reference in their entireties. Bispecific T Cell Engagers (BiTEs)

[0288] An embodiment of the present disclosure includes a bispecific T cell engager (BiTE) that comprises an antigen binding domain and a T cell activation domain.

[0289] In some embodiments, the antigen-binding domain specifically binds to a target antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the antigen-binding domain is any one of the ABPs described herein.

[0290] In certain embodiments, the antigen binding domain is an extracellular antigen binding domain. In certain embodiments, the antigen binding domain specifically binds to CD63. In certain embodiments, the antigen binding domain specifically binds to CD151. In certain embodiments, the antigen binding domain specifically binds to CD72. In certain embodiments, the antigen binding domain specifically binds to CD84. In certain embodiments, the antigen binding domain specifically binds to CD69. In certain embodiments, the antigen binding domain specifically binds to CD109.

[0291] One example of a BiTE is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain) and one or more T cell activation domains. Upon antigen engagement, the intracellular signaling portion of the BiTE can initiate an activation-associated response with T cell-specific molecules, thereby stimulating T cell activation, tumor killing and / or cytokine production.

[0292] In some embodiments, a BiTE comprises an antigen-binding domain that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0293] In some embodiments, a bispecific T cell engager (BiTE) may contain two scFvs produced as a single polypeptide chain. In certain embodiments, a BiTE comprises two scFvs of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109). Methods of making and using BiTE antibodies have been described in the art. See, e.g., Cioffi et al., Clin Cancer Res 18: 465; Brischwein et al., Mol Immunol 43:1129-43 (2006); Amann M et al., Cancer Res 68:143-51 (2008); Schlereth et al., Cancer Res 65: 2882-2889 (2005); and Schlereth et al., Cancer Immunol Immunother 55:785-796 (2006); Huehls A., et al., Immunol Cell Biol. 93(3): 290-296 (2014); Wang et al., Antibodies. 8(32): 1-30 (2019), which are hereby incorporated by reference in their entireties.

[0294] In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0295] In some embodiments, the T cell activation domain comprises a single chain variable fragment (scFV) of an antibody that specifically binds to CD3. In some embodiments, the T cell activation domain comprises the intracellular domain of CD3ζ. In some embodiments, the T cell activation domain comprises a ZAP-70 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0296] In a non-limiting example in which the antigen binding domain and the T cell activation domain both comprise scFvs, the scFvs can be generated by joining the heavy and light chains of each Fv with a serine-glycine linker sequence.

[0297] In certain embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, or six or more GGGGS repeats ("GGGGS" disclosed as SEQ ID NO:2). In certain embodiments, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO:2).

[0298] In certain embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more SGGGG repeats ("SGGGG", disclosed as SEQ ID NO:3). In certain embodiments, the linker comprises the amino acid sequence of SGGGG (SEQ ID NO:3).

[0299] In certain embodiments, the linker may render the peptide long and flexible enough to allow the heavy and light chains to associate in the normal conformation.

[0300] In some embodiments, the linker is a rigid linker, a cleavable linker, or a flexible linker.

[0301] In certain embodiments, the BiTE comprises a linker connecting the antigen-binding domain and the T cell activation domain. As a non-limiting example, when the antigen-binding domain and the T cell activation domain both comprise scFvs, a GGGGS (SEQ ID NO: 2) repeat linker can connect the two scFvs.

[0302] In some embodiments, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 2). In certain embodiments, the linker comprises the amino acid sequence motif (G4S) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 4). In some embodiments, the linker comprises an amino acid sequence of SGGGG (SEQ ID NO: 3). In certain embodiments, the linker comprises an amino acid sequence motif (SG4) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 5). In some embodiments, a linker connects two scFvs. In some embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more GGGGS repeats ("GGGGS" disclosed as SEQ ID NO: 2).

[0303] In some embodiments, the linker comprises 1-25 amino acids. In some embodiments, the linker comprises 1-20 amino acids. In some embodiments, the linker comprises 1-15 amino acids. In certain embodiments, the linker comprises 1-10 amino acids. In certain embodiments, the linker comprises 1-9 amino acids. In certain embodiments, the linker comprises 1-8 amino acids. In certain embodiments, the linker comprises 1-7 amino acids. In some embodiments, the linker comprises 1-6 amino acids. In certain embodiments, the linker comprises 1-5 amino acids. In certain embodiments, the linker comprises 1-4 amino acids. In some embodiments, the linker comprises 1-2 amino acids. In certain embodiments, the linker comprises 1-10 amino acids. In certain embodiments, the length of the linker may determine the flexibility of movement between the two scFvs and may be adjusted by including more or fewer linker repeats to optimize binding to both target cells. For example, in some embodiments, a short, flexible linker connecting the two scFvs may provide free rotation between the antigen-binding domain and the T cell activation domain.

[0304] In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 7).

[0305] Non-limiting examples of linkers can be found in Chen et al., (Chen X., Zaro JL, Shen WC Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. 2013;65:1357-1369), which is hereby incorporated by reference in its entirety.

[0306] In certain embodiments, the linker is selected from the following: (GGGGS)3 (SEQ ID NO: 8), (G) 8 (SEQ ID NO: 9), (G) 6 (SEQ ID NO: 10), (EAAAK) 3 (SEQ ID NO: 11), (EAAAK) n (SEQ ID NO: 12), wherein n is 1 to 3; A(EAAAK) 4 ALEA(EAAAK) 4 A (SEQ ID NO: 13), PAPAP (SEQ ID NO: 14), AEAAAKEAAAKA (SEQ ID NO: 15), (Ala-Pro) n (wherein n is 5 to 17) (SEQ ID NO: 16), VSQTSKLTR↓AETVFPDV b (SEQ ID NO: 17), PLG↓LWA c (SEQ ID NO: 18), RVL↓AEA (SEQ ID NO: 19); EDVVCC↓SMSY (SEQ ID NO: 20), GGIEGR↓GS c (SEQ ID NO:21), TRHRQPR↓GWE (SEQ ID NO:22), and AGNRVRR↓SVG (SEQ ID NO:23). a Protease-sensitive cleavage sites are indicated by “↓”; b factor XIa / FVIIa-sensitive cleavage; c Matrix metalloproteinase-1 sensitive cleavage sequence, one example of which is provided herein; d HIV PR (HIV-1 protease); NS3 protease (HCV protease); factor Xa-sensitive cleavage, respectively; e furin-sensitive cleavage; f Cathepsin B-sensitive cleavage.

[0307] Embodiments of the disclosure include host cells containing BiTEs.

[0308] In some embodiments, the antigen binding domain binds to an epitope on CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the antigen binding domain comprises the VDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. H and VL In some embodiments, the antigen binding domain comprises a CD63, CD151, CD72, CD84, CD69, or CD109 single chain variable fragment (scFv).

[0309] Aspects of the present disclosure include a polynucleotide that encodes BiTE.In some embodiments, the polynucleotide further comprises a sequence that is homologous to the target genome region for site-specific integration.In some embodiments, the polynucleotide is designed for gene editing using endonucleases, such as CRISPR-Cas system, zinc finger nuclease, transcription activator-like effector nuclease (TALEN) and meganuclease.

[0310] The embodiment of the present disclosure includes a vector that comprises polynucleotide. Any vector that can be used for gene delivery can be used. In some variations, viral vectors (e.g., AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, murine stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; and VV, vaccinia virus.

[0311] In some embodiments, the vector is a recombinant AAV vector.In some embodiments, the vector for use in the method of the present disclosure is encapsidated in viral particles (for example, AAV viral particles, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16).

[0312] In some embodiments, the method comprises administering a vector comprising a polynucleotide encoding a BiTE, or a pharmaceutical composition thereof. For example, T cells can be modified using viral or non-viral vectors to facilitate specific targeting of blast cells via expression of an exogenous BiTE. In some embodiments, the vector is used in vitro to generate immunoresponsive cells expressing a BiTE.

[0313] In some embodiments, the methods include administering a vector comprising a polynucleotide encoding a BiTE, or a pharmaceutical composition thereof.

[0314] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide, or a pharmaceutical composition thereof, hi some embodiments, the non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0315] Non-limiting examples of non-viral delivery methods that can be used in the methods of the invention to deliver a polynucleotide include, but are not limited to, physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical vectors, inorganic particles, calcium phosphate particles, magnetic particles, polymer-based vectors, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-co-glycoside) (PLGA), dendrimers, or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06) and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are hereby incorporated by reference in their entireties. Chimeric Antigen Receptors (CARs)

[0316] An embodiment of the present disclosure includes a chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain, a transmembrane domain, a signaling domain, and optionally a costimulatory domain.

[0317] In some embodiments, the extracellular antigen-binding domain specifically binds to a target antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In certain embodiments, the antigen-binding domain specifically binds to CD63. In certain embodiments, the antigen-binding domain specifically binds to CD151. In certain embodiments, the antigen-binding domain specifically binds to CD72. In certain embodiments, the antigen-binding domain specifically binds to CD84. In certain embodiments, the antigen-binding domain specifically binds to CD69. In certain embodiments, the antigen-binding domain specifically binds to CD109.

[0318] In some embodiments, the antigen binding domain is any one of the ABPs described herein.

[0319] One example of a CAR is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen engagement, the intracellular signaling portion of the CAR can initiate an activation-related response in immune cells, such as the release of cytolytic molecules to induce tumor cell death.

[0320] In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, a signaling domain, and optionally a costimulatory domain, wherein the extracellular antigen-binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In certain embodiments, the CAR further comprises a polypeptide hinge region.

[0321] In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0322] In some embodiments, the signaling domain comprises the intracellular domain of CD3zeta. In some embodiments, the signaling domain comprises a ZAP-70 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0323] In some embodiments, the co-stimulatory domain further comprises one or more (e.g., two or more, three or more, four or more, or five or more) co-stimulatory domains, which are CD28, 4-1BB, CD27, OX40, or ICOS co-stimulatory domains. In some embodiments, the co-stimulatory domain is selected from 4-1BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0324] In some embodiments, the CAR comprises 4-1BB, CD28 or a fragment thereof. In certain embodiments, the CAR comprises a CD28 transmembrane domain and 4-1BB. In certain embodiments, the CAR comprises a hinge region, a CD28 transmembrane domain and 4-1BB. In certain embodiments, the hinge region is derived from a CD28 polypeptide.

[0325] In some embodiments, the CAR comprises a hinge region of a CD28 polypeptide, wherein the hinge region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 99).

[0326] In some embodiments, the CAR comprises a transmembrane domain of a CD28 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 100).

[0327] In some embodiments, the CD28 costimulatory domain comprises: [ka] The amino acid sequence of the present invention has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of the present invention.

[0328] In some embodiments, the zeta (CD3ζ) signaling domain is [ka] The amino acid sequence of the present invention has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of the present invention.

[0329] In some embodiments, the CAR comprises a CD28 transmembrane (TM) and 4-1BB costimulatory domain in combination with a zeta (CD3ζ) signaling domain.

[0330] In some embodiments, the CAR construct is placed in a plasmid, for example, a pUC57 plasmid. Using restriction enzyme techniques, the coding sequence of the CAR can be excised and placed in a vector transfer plasmid for high titer viral vector production (see, for example, Figures 13 and 14).

[0331] In some embodiments, the CAR comprises a signal peptide (e.g., a signal interfering peptide (SIP)). In certain embodiments, the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 103). In certain embodiments, the signal peptide is [ka] The nucleic acid is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of

[0332] In some embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises an scFv of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, and / or CD109). In certain embodiments, the scFv binds to the target protein CD63. In certain embodiments, the scFv binds to the target protein CD151. In certain embodiments, the scFv binds to the target protein CD72. In certain embodiments, the scFv binds to the target protein CD84. In certain embodiments, the scFv binds to the target protein CD69. In certain embodiments, the scFv binds to the target protein CD109. In some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 89-98 of Table 12.

[0333] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:89.

[0334] In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:89 above.

[0335] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:90.

[0336] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:91.

[0337] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:92.

[0338] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:93.

[0339] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:94.

[0340] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:95.

[0341] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:96.

[0342] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:97.

[0343] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the scFv comprises a nucleotide sequence encoding an scFv region, wherein the nucleotide sequence encodes an scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:98.

[0344] In some embodiments, the CAR comprises a signal peptide, an scFv region, one or more costimulatory domains, and a signaling domain.

[0345] In order of appearance, Table 14 provides the nucleotide sequences of the CAR constructs (SEQ ID NOs:24-34), and Table 14 provides the protein sequences of the CAR constructs (SEQ ID NOs:35-44 and 36), respectively.

[0346]

[0347] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-44 of Table 13 provided in Table 13.

[0348] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0349] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 24 in Table 14.

[0350] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 25 in Table 14.

[0351] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 26 in Table 14.

[0352] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 27 in Table 14.

[0353] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 28 in Table 14.

[0354] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:40 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:29 in Table 14.

[0355] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:41 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:30 in Table 14.

[0356] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:42 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:31 in Table 14.

[0357] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:43 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:32 in Table 14.

[0358] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:44 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:33 in Table 14.

[0359] In some embodiments, a CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36 in Figure 27. In some embodiments, a CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 34 in Table 14.

[0360] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8]

[0361]

[0362] Aspects of the present disclosure include a host cell comprising a CAR. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a CD4 + and CD8 + In certain embodiments, the host cells are naive cell memory (T n + In some embodiments, the host cells are stem cell memory (T scm In some embodiments, the host cells are stem cell memory ((T cm )CD197[CCR7 + ] + CD45R0 - In some embodiments, the host cells are central memory (T cm , CD197(CCR7) + CD45R0 + ) T cells. CD197 is used interchangeably with "CCR7" herein.

[0363] Aspects of the present disclosure include one or more host cells comprising a CAR. In certain embodiments, the one or more host cells comprising a CAR are naïve and stem cell memory (T n + T scm , CD197[CCR7 + ] + CD45R0 - ), and Central Memory (T cm , CD197[CCR7] + CD45R0 + ) T cells. In some embodiments, the one or more cells comprising a CAR are natural killer (NK) cells.

[0364] In some embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain and a signal transduction domain.In some embodiments, the CAR is a second generation CAR that comprises an extracellular antigen binding domain, a transmembrane domain, a signal transduction domain and a costimulatory domain.In some embodiments, the CAR comprises an extracellular antigen binding domain, a hinge region of a polypeptide, a transmembrane domain and a signal transduction domain.

[0365] In some embodiments, the extracellular antigen binding domain binds to an epitope on a CD63, CD151, CD72, CD84, CD69, or CD109 target antigen. In some embodiments, the extracellular antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the extracellular antigen binding domain comprises the VDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. H and V L In some embodiments, the extracellular antigen-binding domain comprises a CD63, CD151, CD72, CD84, CD69, or CD109 single chain variable fragment (scFv).

[0366] Aspects of the present disclosure include a polynucleotide that encodes a CAR.In some embodiments, the polynucleotide further comprises a sequence that is homologous to the target genome region for site-specific integration.In some embodiments, the polynucleotide is designed for gene editing using endonucleases, such as CRISPR-Cas system, zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and meganucleases.

[0367] The embodiment of the present disclosure includes a vector that comprises polynucleotide. Any vector that can be used for gene delivery can be used. In some variations, viral vectors (e.g., AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, murine stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; and VV, vaccinia virus.

[0368] In some embodiments, the vector is a lentiviral vector. Lentiviruses are a subclass of retroviruses. However, lentiviruses can integrate into the genome of non-dividing cells, whereas retroviruses can only infect dividing cells.

[0369] Lentiviral vectors are typically produced from a packaging cell line, typically HEK293, that has been transformed with several plasmids, including (1) a packaging plasmid that encodes the virion proteins, e.g., capsid and reverse transcriptase, and (2) a plasmid that contains the exogenous gene to be delivered to the target.

[0370] When virus invades cell, viral genome in the form of RNA is reverse transcribed to produce DNA, and this DNA is then inserted into genome by viral integrase enzyme.Therefore, the exogenous substance delivered by lentivirus vector can remain in genome and be transmitted to cell progeny when cell divides.

[0371] In some embodiments, the vector is a recombinant AAV vector.In some embodiments, the vector for use in the method of the present disclosure is encapsidated in viral particles (for example, AAV viral particles, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16).

[0372] Adeno-associated virus can infect non-dividing cells and various types of cells, which makes it useful in constructing the gene delivery system of the present disclosure.Detailed description of the use and preparation of AAV vectors can be found in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368.

[0373] Research results regarding AAV as a gene delivery system are disclosed in LaFace et al, Biology, 162: 483486 (1988), Zhou et al., Exp. Hematol. (NY), 21:928-933(1993), Walsh et al, J. Clin. Invest., 94:1440-1448(1994) and Flotte et al., Gene Therapy, 2:29-37(1995). Typically, recombinant AAV viruses are generated by cotransfecting a plasmid containing the gene of interest (i.e., the decorin gene and the nucleotide sequence of interest to be delivered) flanked by two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989) and an expression plasmid containing the wild-type AAV coding sequence without the terminal repeats (McCarty et al., J. Viral., 65:2936-2945(1991)).

[0374] In some embodiments, the method includes administering a vector comprising a polynucleotide encoding a CAR, or a pharmaceutical composition thereof. For example, T cells can be modified using viral or non-viral vectors to facilitate specific targeting of blast cells via expression of an exogenous CAR. In some embodiments, the vector is used in vitro to generate an immunoresponsive cell expressing a CAR (e.g., a CAR-T cell). Aspects of the present disclosure include an immunoresponsive cell expressing a CAR. Aspects of the present disclosure include an immunoresponsive cell comprising a polynucleotide encoding a CAR or a vector comprising a polynucleotide.

[0375] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide, or a pharmaceutical composition thereof, hi some embodiments, the non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0376] Non-limiting examples of non-viral delivery methods that can be used in the methods of the invention to deliver a polynucleotide include, but are not limited to, physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical vectors, inorganic particles, calcium phosphate particles, magnetic particles, polymer-based vectors, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-co-glycoside) (PLGA), dendrimers, or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06) and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are hereby incorporated by reference in their entireties.

[0377] In some embodiments, the immunoresponsive cell is a bispecific CAR-T that targets two target proteins selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the immunoresponsive cell is a bispecific CAR-T that targets (i) one target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; and (ii) CD3. In some embodiments, the immunoresponsive cell is a bispecific CAR-T that targets (i) one target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; and (ii) CD123.

[0378] In some embodiments, the immunoresponsive cells are αβ T cells, γδ T cells, or natural killer (NK) cells. + T cells, or CD4 + T cells or CD8 + T cells.

[0379] Aspects of the present disclosure include a method for preparing an immunoresponsive cell. In some embodiments, the method comprises transfecting or transducing a polynucleotide encoding a CAR or a vector containing a polynucleotide encoding a CAR into the immunoresponsive cell. In some embodiments, the method comprises expanding the immunoresponsive cell for at least 48 hours. In some embodiments, the immunoresponsive cell is transduced at a multiplicity of infection of at least 10.

[0380] In some embodiments, the method further comprises, after the transducing step, washing the vector and expanding the CAR T cells for at least 14 days.

[0381] Transduction efficiency can be measured by digital droplet PCR (ddPCR) and expressed as vector copy number (VCN) per cell. In some embodiments, the immunoresponsive CAR T cells express a CAR T cell marker in the range of 2.5 to 33.5 (e.g., 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, Includes average VCNs for: 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31.5, 32, 32.5, 33, 33.5, etc.

[0382] In some embodiments, after transducing a polynucleotide encoding a CAR or a vector containing a polynucleotide encoding a CAR into an immunoresponsive cell, the method comprises measuring the rate of expansion of the immunoresponsive cell. In certain embodiments, the rate of expansion of the immunoresponsive cell is between 2-fold and 20-fold (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-fold).

[0383] In some embodiments, the immunoresponsive cells include naïve and stem cell memory (T n +T scm , CD197[CCR7] + CD45R0 - ), and / or Central Memory (T cm , CD197(CCR7) + CD45R0 + ) T cells.

[0384] In some embodiments, immunoresponsive CAR T cells show both activation and exhaustion to the same extent as empty CAR-T cells.This can be shown by the upregulation of CD25, CD154, CD107a and CD137, and the upregulation of CD366, CD223 and CD279.In certain embodiments, the immunoresponsive cells containing CAR do not change T cell function. 3.4. Diagnostic methods

[0385] In one aspect, the present disclosure provides a method for diagnosing cancer.In particular, the method can be used to diagnose myeloid disorder or acute leukemia in a subject.The method includes detecting the presence or absence or level of tumor-specific antigen in a biological sample of a subject, and the tumor-specific antigen is selected from CD63, CD151, CD72, CD84, CD69 and CD109.

[0386] In some embodiments, the detecting step comprises contacting the biological sample with an ABP, the ABP specifically binding to the tumor-specific antigen. In some embodiments, the ABP is an antibody or antigen-binding fragment that binds to the tumor-specific antigen.

[0387] ABPs that can be used for the detecting step are described in Section 3.3.1 "Antigen Binding Proteins (ABPs)."

[0388] In some embodiments, the detecting step comprises flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the ABP is labeled. In some embodiments, the ABP is labeled with a fluorophore or enzyme.

[0389] In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or next-generation sequencing.

[0390] In some embodiments, the method further comprises a step of treatment based on the diagnosis. Biological samples

[0391] In some embodiments, the biological sample is a blood sample from the subject. In certain embodiments, the blood sample is a peripheral blood sample. In some embodiments, the biological sample is a bone marrow sample. In some embodiments, the biological sample comprises a solid or liquid tumor. In some embodiments, the biological sample is an AML patient-derived xenograft (PDX).

[0392] In some embodiments, the biological sample comprises a blast cell. In certain embodiments, the blast cell is selected from a myeloid blast (e.g., a myeloblast), a lymphoid blast, or a combination of myeloid and lymphoid blast cells. In some embodiments, the blast cell is a myeloid blast cell. In some embodiments, the blast cell is a lymphoid blast cell. In some embodiments, the blast cell is a combination of myeloid and lymphoid blast cells. In some embodiments, the biological sample is an AML blast cell. 3.4.2.Detecting the Presence or Level of TSA

[0393] In some embodiments, the method comprises detecting the presence / absence or level of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or level of two TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or level of three TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or level of four TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or level of five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or level of six TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109.

[0394] In some embodiments, the detecting step comprises contacting the biological sample with an ABP. In some embodiments, the ABP specifically binds to one of the tumor-specific antigens. In some embodiments, the ABP is an anti-CD63 antibody. In some embodiments, the ABP is an anti-CD151 antibody. In some embodiments, the ABP is an anti-CD72 antibody. In some embodiments, the ABP is an anti-CD84 antibody. In some embodiments, the ABP is an anti-CD69 antibody. In some embodiments, the ABP is an anti-CD109 antibody.

[0395] In some embodiments, the ABP is labeled. In some embodiments, the ABP is labeled with a fluorophore or an enzyme. In some embodiments, the ABP is labeled with a radioisotope. In some embodiments, the ABP is coupled to alkaline phosphatase, horseradish peroxidase, beta-galactosidase, tobacco etch virus nuclear-inclusion-a endopeptidase ("TEV protease"). In some embodiments, the ABP is 1,8-ANS, 4-Methylumbelliferone, 7-Amino-4-methylcoumarin, 7-Hydroxy-4-methylcoumarin, Acridine, Alexa Fluor 350™, Alexa Fluor 405™, AMCA, AMCA-X, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTO Rho101, Pacific Blue, Alexa Fluor 43Q™, Alexa Fluor480™, Alexa Fluor488™, BODIPY 492 / 515, Alexa Fluor 532™, Alexa Fluor 546™, Alexa Fluor555™, Alexa Fluor594™, BODIPY 505 / 515, Cy2, cyQUANT GR, FITC, Fluo-3, Fluo-4, GFP (EGFP), mHoneydew, Oregon Green™ 488, Oregon Green™ 514, EYFP, DsRed, DsRed2, dTomato, Cy3.5, Phycoerythrin (PE), Rhodamine Red, mTangerine, mStrawberry, mOrange, mBanana, Tetramethylrhodamine (TRITC), R-Phycoerythrin, ROX, DyLight 594, Calcium Crimson, Alexa Fluor594™, Alexa Fluor610™, Texas Red, mCherry, mKate, Alexa Fluor660™, Alexa Fluor680™, Allophycocyanin, DRAQ-5, Carboxynaphthofluorescein, C7, DyLight 750, Cellvue NIR780, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDye (IRD40, IRD 700, IRD 800), JOE, Lissamine, Rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, PyMPO, 5-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 5-Carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylamino, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, dansyl chloride, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, europium trisbipyridinediamine In some embodiments, the ABP is coupled to a fluorophore selected from the group consisting of europium cryptates or chelates, diamines, dicyanins, and La Jolla blue dyes. In some embodiments, the ABP is conjugated to a quantum dot.

[0396] In some embodiments, the detecting step comprises flow cytometry, immunohistochemistry, immunofluorescence, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the detecting step comprises Western blotting. In some embodiments, the detecting step comprises mass spectrometry.

[0397] In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample. In certain embodiments, the method comprises measuring the mRNA level of CD63. In certain embodiments, the method comprises measuring the mRNA level of CD151. In certain embodiments, the method comprises measuring the mRNA level of CD72. In certain embodiments, the method comprises measuring the mRNA level of CD84. In certain embodiments, the method comprises measuring the mRNA level of CD69. In certain embodiments, the method comprises measuring the mRNA level of CD109.

[0398] In some embodiments, mRNA levels are measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), or sequencing (eg, next-generation sequencing).

[0399] In some embodiments, the method further comprises determining the presence or absence of cancer in the subject based on the presence / absence or level of TSA in a sample from the subject.

[0400] In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) if it exhibits expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69, and CD 109. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) if it exhibits expression of at least two, three, four, or five TSAs selected from CD63, CD151, CD72, CD84, CD69, and CD109.

[0401] In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when it shows expression of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when it shows expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when it shows expression of CD63, CD151, CD72, CD84, CD69 and CD109.

[0402] In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when the subject shows increased expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to a control sample. In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when the subject shows increased expression of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to a control sample. In some embodiments, a subject is diagnosed with myeloid disorder (MD) or acute leukemia (AL) when the subject shows increased expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to a control sample. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) if the subject exhibits increased expression of CD63, CD151, CD72, CD84, CD69 and CD109 compared to a control sample.

[0403] In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69, CD109, and additionally at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine TSAs selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR, compared to a control sample. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) if the subject exhibits increased expression of one TSA selected from CD63, CD151, CD72, CD84, CD69, CD109, and additionally at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine TSAs selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR, compared to a control sample. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69, CD109, and additionally at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine TSAs selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3 and HLA-DR, compared to a control sample. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) if the subject exhibits increased expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine TSAs selected from CD63, CD151, CD72, CD84, CD69, CD109, and also CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR, as compared to a control sample.

[0404] In one embodiment, a subject is diagnosed with MD or AL if it shows at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, or at least a 90% increase in expression of TSA compared to a predefined reference level of TSA or compared to a sample from a healthy subject. In some embodiments, a subject is diagnosed with MD or AL if it shows at least a 2, 3, 4, 5, 10, 20, 50, or 100-fold increase in expression of TSA compared to a predefined reference level of TSA or compared to a sample from a healthy subject.

[0405] In some embodiments, the method further comprises determining the presence or absence of cancer in the subject based on detecting the presence or level of a tumor-specific antigen in a sample from the subject. 3.4.3. Myeloid disorders and acute leukemia

[0406] In some embodiments, the myeloid disorder is a myeloid malignancy. In some embodiments, the myeloid disorder is myeloid leukemia. In certain embodiments, the myeloid leukemia is acute myeloid leukemia (AML). In certain embodiments, the myeloid leukemia is childhood acute myeloid leukemia.

[0407] In some embodiments, the myeloid disorder is a myeloid neoplasm. In some embodiments, the myeloid disorder is selected from myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic / myeloproliferative neoplasms (MDS / MPN), and myeloid malignancies associated with eosinophilia and abnormalities in growth factor receptors derived from platelets or fibroblasts.

[0408] In some embodiments, the MDS is selected from refractory cytopenia with single lineage dysplasia (refractory anemia; refractory neutropenia, refractory thrombocytopenia), refractory anemia with ring siderblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts-1, refractory anemia with excess blasts-2, myelodysplastic syndrome with isolated 5q deletion, and myelodysplastic syndrome. In some embodiments, the MPN is selected from chronic myelogenous leukemia, polycythemia vera, essential thombocythemia, primary myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia, hypereosinophilic syndrome, and mast cell disease. In some embodiments, the MDS / MPN is selected from chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, and atypical chronic myelodysplastic leukemia. In some embodiments, the myeloid neoplasm is selected from PDGFRA rearrangement-associated myeloid neoplasms, PDGFRB rearrangement-associated myeloid neoplasms, and FGFR1 rearrangement-associated myeloid neoplasms (e.g., 8p11 myeloproliferative syndrome). In some embodiments, the acute leukemia is selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), lymphocytic leukemia (LL), myeloid leukemia (ML).

[0409] In some embodiments, the myeloid disorders and acute leukemias have an onset in pediatric or adult age. In some embodiments, the myeloid disorders and acute leukemias are pediatric AML. In certain embodiments, the myeloid disorders and acute leukemias have an onset in subjects between 1 year and 18 years old. In certain embodiments, the myeloid disorders and acute leukemias have an onset in subjects between 1 day and 18 years old. In certain embodiments, the myeloid disorders and acute leukemias have an onset in subjects between 1 day and 1 year old.

[0410] In some embodiments, myeloid disorder and acute leukemia is adult AML.In certain embodiments, myeloid disorder and acute leukemia have onset in adults older than 18 years old, older than 20 years old, older than 25 years old, older than 30 years old, older than 35 years old, older than 40 years old, older than 45 years old, older than 50 years old, older than 55 years old, older than 60 years old or older than 65 years old. 3.5. Treatment Method

[0411] In another aspect, the disclosure provides a method of treating a subject with a hematological malignancy. In some embodiments, the subject has refractory disease. In some embodiments, the subject has a relapse.

[0412] In certain embodiments, the hematological malignancy is a refractory B-cell malignancy. B-cell malignancies include B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia / small lymphocytic lymphoma, monoclonal B-cell lymphocytosis, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic B-cell lymphoma / leukemia-unclassified, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, Waldenstrom's hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) IgM, m heavy chain disease, g heavy chain disease, a heavy chain disease, MGUS IgG / A, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, monoclonal immunoglobulin deposition disease, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, follicular lymphoma, follicular neoplasm in situ, duodenal follicular lymphoma, pediatric follicular lymphoma, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, mantle cell lymphoma, mantle cell neoplasm in situ, diffuse large B-cell lymphoma (DLBCL) NOS including germinal center B-cell type and activated B-cell type; T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL, leg type, EBV+ DLBCL These include, but are not limited to, NOS, EBV+ mucocutaneous ulcer, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8+ DLBCL NOS, Burkitt lymphoma, Burkitt-like lymphoma with llq aberrations, high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements, high-grade B-cell lymphoma NOS, and B-cell lymphoma, unclassifiable type with features intermediate between DLBCL and classical Hodgkin lymphoma.In some embodiments, the malignant disease treated with the anti-CD72 nanobodies described herein is Hodgkin lymphoma, e.g., nodular lymphocyte-predominant Hodgkin lymphoma, or classical Hodgkin lymphoma, including nodular sclerosing classical Hodgkin lymphoma, lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, and lymphocyte-depleted classical Hodgkin lymphoma. In some embodiments, the subject has post-transplant lymphoproliferative disorder (PTLD), e.g., plasmacytic hyperplasia PTLD, infectious mononucleosis PTLD, follicular hyperplasia PTLD, polymorphic PTLD, monomorphic PTLD (B and T / NK cell type), or classical Hodgkin lymphoma PTLD.

[0413] In one aspect, the disclosure provides a method of treating a subject having a hematological malignancy, such as a malignancy involving malignant B cells or malignant myeloid cells. In some embodiments, the hematological malignancy is B cell leukemia. In some embodiments, the B cell leukemia is chronic lymphocytic leukemia. In some embodiments, the B cell leukemia is mixed lineage leukemia (MLL). In some embodiments, the hematological malignancy is non-Hodgkin's lymphoma. In some embodiments, the hematological malignancy is multiple myeloma. In some embodiments, the hematological malignancy comprises myeloid cells expressing any one of the target proteins selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the hematological malignancy comprises one or more, two or more, three or more, four or more, or five or more of the target proteins.

[0414] In one aspect, the present disclosure provides a method for treating a subject with myeloid disorder (MD) or acute leukemia (AL). In some embodiments, the acute leukemia is acute lymphoblastic leukemia.

[0415] In some embodiments, the subject has AML of the myeloblastic (M0) type. In some embodiments, the subject has AML of the myeloblastic (M0) type. In some embodiments, the subject has AML of the myeloblastic (M1) type. In some embodiments, the subject has AML of the myeloblastic (M2) type. In some embodiments, the subject has AML of the promyelocytic (M3) type. In some embodiments, the subject has AML that is AML of the myelomonocytic (M4) type. In some embodiments, the subject has AML that is AML of the monocytic (M5) type. In some embodiments, the subject has AML that is AML of the erythroleukemia (M6) type. In some embodiments, the subject has AML that is AML of the megakaryocytic (M7) type.

[0416] In some embodiments, the subject includes one or more, two or more, three or more, four or more, or five or more of the target proteins.

[0417] In some embodiments, the methods of the disclosure include administering to a subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD 109. The therapeutic agent can be an antigen binding protein (ABP) described herein, an ABP-drug conjugate, a bispecific T cell engager (BiTE), or an immunoresponsive cell expressing a chimeric antigen receptor (CAR) specific for the target protein.

[0418] In certain embodiments, the therapeutic agent is a CAR-T cell. In certain embodiments, the therapeutic agent is a CAR-NK cell. In certain embodiments, the CAR-T cell or CAR-NK cell does not cause hematologic toxicity after administration to a subject. In some embodiments, the method of treatment with CAR-T cell or CAR-NK cell provided herein does not require supportive care. In some embodiments, the method includes administering a therapeutically effective amount of an immunoresponsive cell comprising a CAR, and the administering step is not followed or performed in combination with immunoglobulin therapy or autologous / allogeneic HSC to support hematopoiesis. In some embodiments, the immunoglobulin therapy is intravenous immunoglobulin (IVIG) treatment.

[0419] In some embodiments, the ABP or pharmaceutical composition is not administered in combination with an immunotherapy. In some embodiments, the ABP or pharmaceutical composition is not administered by combination therapy. In some embodiments, the ABP or pharmaceutical composition is not administered by immunotherapy. In some embodiments, the method comprises administering a therapeutically effective amount of an immunoresponsive cell comprising a CAR, and the administering step is not followed or combined with autologous or allogeneic hematopoietic stem cell therapy to support hematopoiesis.

[0420] In some embodiments, the method further comprises treating the subject with a chemotherapeutic agent or hematopoietic stem cells prior to administering the ABP, pharmaceutical composition or immunoresponsive cell. In some embodiments, the subject has a refractory disease. In some embodiments, the subject has a refractory cancer. In some embodiments, the subject has a relapse. In some embodiments, the subject is not responsive to treatment with a chemotherapeutic agent or hematopoietic stem cell transplant.

[0421] In some embodiments, the therapeutic agent is administered in an amount sufficient to affect the survival of cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In certain embodiments, the therapeutic agent is administered in an amount sufficient to eliminate cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to reduce or kill cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to reduce or kill cancer cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to inhibit a biological effect associated with CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to affect the survival of cancer cells in the subject.

[0422] In some embodiments, the method of the present disclosure comprises administering to a subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, the treatment being a first treatment (e.g., a first treatment or a first line therapy) administered to the subject for the treatment of a hematological malignancy. In some embodiments, the method of the present disclosure comprises administering to a subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, the treatment being a second treatment (e.g., a second treatment or a second line therapy) administered to the subject for the treatment of a hematological malignancy. For example, in certain embodiments, the subject has undergone a first treatment for treating a hematological malignancy prior to treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. In certain embodiments, the first treatment is selected from one or more of chemotherapy and hematopoietic stem cell transplantation treatment.

[0423] In some embodiments, administering to a subject a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109 is a first-line therapy. In some embodiments, administering to a subject a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109 is a second-line therapy. In certain embodiments, administering to a subject a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109 is a first-line therapy. In certain embodiments, administering to a subject a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109 is not combined with any other cancer treatment.

[0424] In some embodiments, the subject has refractory disease before treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.In certain embodiments, the subject is a subject before treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, and is not responsive to cancer treatment.In certain embodiments, the subject is a relapsed subject in which cancer cells are present in the patient after cancer treatment before treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.In certain embodiments, the subject has not responded to first-line therapy before treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. In certain embodiments, the patient is non-responsive to first line therapy prior to treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0425] In some embodiments, the subject is an adult.In certain embodiments, the subject is an adult that is older than 18 years old, older than 20 years old, older than 25 years old, older than 30 years old, older than 35 years old, older than 40 years old, older than 45 years old, older than 50 years old, older than 55 years old, older than 60 years old, or older than 65 years old.In certain embodiments, the subject is a pediatric subject.In certain embodiments, the subject is younger than 18 years old, younger than 20 years old, younger than 25 years old, younger than 30 years old, younger than 35 years old, younger than 40 years old, younger than 45 years old, younger than 50 years old, younger than 55 years old, younger than 60 years old, or younger than 65 years old.

[0426] Aspects of the present disclosure include polynucleotides that code for ABP, ABP-drug conjugate, bispecific T cell engager (BiTE) or chimeric antigen receptor (CAR). In some embodiments, the polynucleotide further comprises a sequence that is homologous to the target genome region for site-specific integration. In some embodiments, the polynucleotide is designed for gene editing using endonucleases, such as CRISPR-Cas system, zinc finger nuclease, transcription activator-like effector nuclease (TALEN) and meganuclease.

[0427] The embodiment of the present disclosure includes a vector that comprises polynucleotide. Any vector that can be used for gene delivery can be used. In some variations, viral vectors (e.g., AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, murine stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; and VV, vaccinia virus.

[0428] In some embodiments, the vector is a lentiviral vector. Lentiviruses are a subclass of retroviruses. However, lentiviruses can integrate into the genome of non-dividing cells, whereas retroviruses can only infect dividing cells.

[0429] In some embodiments, the vector is a recombinant AAV vector.In some embodiments, the vector for use in the method of the present disclosure is encapsidated in viral particles (for example, AAV viral particles, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16).

[0430] In some embodiments, the method comprises administering a vector comprising the polynucleotide, or a pharmaceutical composition thereof, hi some embodiments, the vector is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0431] In some embodiments, the method comprises administering a non-viral vector comprising a polynucleotide encoding a therapeutic agent, or a pharmaceutical composition thereof, hi some embodiments, the non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0432] Non-limiting examples of non-viral delivery methods that can be used in the methods of the invention to deliver a polynucleotide encoding an ABP include, but are not limited to, physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical vectors, inorganic particles, calcium phosphate particles, magnetic particles, polymer-based vectors, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-co-glycoside) (PLGA), dendrimers, or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06) and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are hereby incorporated by reference in their entireties.

[0433] In some embodiments, the method further comprises detecting the presence / absence or level of a tumor-specific antigen in a biological sample of the subject, as described in Section 3.4, before or after administration of the therapeutic agent. In some embodiments, the treatment methods provided herein are used to treat a subject diagnosed with MD or AL using the methods described in Section 3.4. In some embodiments, the diagnostic methods described in Section 3.4 are used to check the effectiveness of the treatment methods provided herein. In some embodiments, the method comprises determining the presence or absence of myeloid disorder (MD) and acute leukemia (AL) blasts in a sample obtained from the subject.

[0434] In some embodiments, the methods of the disclosure include treating a subject having a myeloid disorder (MD) or acute leukemia (AL) and include administering an effective amount of an ABP, an ABP-drug conjugate, a BiTE, a CAR, or an immunoresponsive cell comprising a CAR, or a pharmaceutical composition thereof.

[0435] In some embodiments, the immunoresponsive cells expressing ABP, ABP-drug conjugate, BiTE, or CAR are administered in an amount sufficient to affect the survival of cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In certain embodiments, the immunoresponsive cells expressing ABP, ABP-drug conjugate, BiTE, or CAR are administered in an amount sufficient to eliminate cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the immunoresponsive cells expressing ABP, ABP-drug conjugate, BiTE, or CAR are administered in an amount sufficient to reduce or kill cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the immunoresponsive cells expressing the ABP, ABP-drug conjugate, BiTE, or CAR are administered in an amount sufficient to reduce or kill cancer cells expressing CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the immunoresponsive cells expressing the ABP, ABP-drug conjugate, BiTE, or CAR are administered in an amount sufficient to reduce or kill cancer cells in the subject.

[0436] In some embodiments, the effective amount of immunoresponsive cells expressing a CAR ranges from 100,000 cells / kg to 15 million cells / kg. In some embodiments, the method includes administering to the subject immunoresponsive cells expressing a CAR at a dose ranging between 100,000 cells / kg to 25 million cells / kg.

[0437] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D In some embodiments, the ABP, ABP-drug conjugate, BiTE or CAR binds to a target protein with a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D The antibody comprises an antigen-binding domain that binds to a target protein.

[0438] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR has a K of less than 500 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by biolayer interferometry. D In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR binds to human CD63, CD151, CD72, CD84, CD69, or CD109 with a K of less than 500 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by biolayer interferometry. D and an antigen-binding domain that binds to human CD63, CD151, CD72, CD84, CD69, or CD109.

[0439] In some embodiments, the ABP, ABP-drug conjugate, BiTE or CAR comprises an antigen-binding domain of a commercially available antibody or a modification thereof obtained by affinity maturation. In some embodiments, the commercially available antibody is an antibody against CD69 (e.g., Invitrogen 14-0699-82, ab201570, R&D MAB2359). In some embodiments, the commercially available antibody is an antibody against CD63 (e.g., ab59479, BD556019). In some embodiments, the commercially available antibody is an antibody against CD151 (e.g., Invitrogen MA5-16443, BD 556056). In some embodiments, the commercially available antibody is an antibody against CD84 (e.g., Biolegend 326002, NOVUS NBP2-44345). In some embodiments, the commercially available antibody is an antibody against CD109 (e.g., R&D MAB4385, BD56019). In some embodiments, the commercially available antibody is an antibody against CD72 (e.g., Biolegend 316202, BD 555917, MAB 5405, Invitrogen PAS-97567).

[0440] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR comprises an antigen-binding domain of A1, C1, F1, G1, C2, F2, H2, H3, F12, or B8, or a modification thereof. 3.5.1. Additional Medications

[0441] In some embodiments, the methods of treatment provided herein further comprise administering an additional agent in combination with the ABP or a pharmaceutical composition thereof, the ABP-drug conjugate or a pharmaceutical composition thereof, the BiTE or a pharmaceutical composition thereof, or the immunoresponsive cell expressing a CAR or a pharmaceutical composition thereof. In some embodiments, the additional agent is administered separately, sequentially, or together with the ABP or a pharmaceutical composition thereof, the ABP-drug conjugate or a pharmaceutical composition thereof, the BiTE or a pharmaceutical composition thereof, or the immunoresponsive cell expressing a CAR or a pharmaceutical composition thereof.

[0442] In some embodiments, the ABP or pharmaceutical composition thereof is administered in combination with an additional agent. In some embodiments, the CAR-expressing immunoresponsive cell or pharmaceutical composition thereof is administered in combination with an additional agent. Administering an additional agent in combination with the ABP or pharmaceutical composition thereof may include administering the additional agent before, during or after administering the ABP or pharmaceutical composition. In some embodiments, administering an additional agent in combination with the ABP or pharmaceutical composition thereof includes administering the additional agent before administering the ABP or pharmaceutical composition thereof. In some embodiments, administering an additional agent in combination with the ABP or pharmaceutical composition thereof includes administering the additional agent after administering the ABP or pharmaceutical composition thereof. In some embodiments, administering an additional agent in combination with the ABP or pharmaceutical composition thereof includes administering the additional agent in parallel with administering the ABP or pharmaceutical composition thereof.

[0443] In some embodiments, the additional agent is a chemotherapeutic agent or a biologic agent.

[0444] In certain embodiments, the additional agent is a chemotherapeutic agent, hi certain embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacytidine, decitabine, and CPX-351 (Vyxeos®).

[0445] In certain embodiments, the chemotherapeutic agent is fludarabine. In other particular embodiments, the chemotherapeutic agent is cyclophosphamide.

[0446] In certain embodiments, the additional agent is a biologic agent, hi some embodiments, the biologic agent is an antibody to a target protein other than CD63, CD151, CD72, CD84, CD69, or CD109.

[0447] In some embodiments, the additional agent is a hedgehog pathway inhibitor. In some embodiments, the hedgehog pathway inhibitor is a sonic hedgehog pathway inhibitor. In some embodiments, the sonic hedgehog pathway inhibitor is selected from vismodegib, sonidegib and arsenic trioxide (ATO). In certain embodiments, the hedgehog pathway inhibitor is glasdegib (Daurismo™).

[0448] In some embodiments, the additional agent is an FMS-like tyrosine kinase 3 (FLT3) inhibitor. In certain embodiments, the FLT3 inhibitor is selected from the group consisting of midostaurin (Rydapt®), gilteritinib (Xospata®), sorafenib, lestaurtinib, quizartinib, and crenolanib.

[0449] In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In certain embodiments, the IDH1 or IDH2 inhibitor is ivosidenib (Tibsovo®) or enasidenib (Idhifa®).

[0450] In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In certain embodiments, the BCL2 inhibitor is venetoclax (Venclexta®).

[0451] In some embodiments, the additional agent is a CD33 targeting agent. In certain embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg™) or vadastuximab butarilin (SGN-CD33A).

[0452] In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor.

[0453] In some embodiments, the additional agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. Pharmaceutical Compositions

[0454] In yet another aspect, the present disclosure provides a pharmaceutical composition containing an immunoresponsive cell expressing an antigen binding protein (ABP), an ABP-drug conjugate, a BiTE, or a chimeric antigen receptor (CAR) described herein. Such a composition comprises an effective amount of an immunoresponsive cell expressing an antigen binding protein (ABP), an ABP-drug conjugate, a BiTE, or a chimeric antigen receptor (CAR) in admixture with a pharma- ceutically acceptable excipient.

[0455] In one aspect, the disclosure includes a pharmaceutical composition comprising an ABP and a pharma- ceutically acceptable excipient.

[0456] In another aspect, the present disclosure includes a pharmaceutical composition comprising an ABP-drug conjugate and a pharma- ceutically acceptable excipient.

[0457] In another aspect, the disclosure includes a pharmaceutical composition comprising a BiTE and a pharma- ceutically acceptable excipient.

[0458] In another aspect, the present disclosure includes a pharmaceutical composition comprising an immunoresponsive cell expressing a chimeric antigen receptor (CAR) and a pharma- ceutically acceptable excipient.

[0459] Pharmaceutical compositions may contain formulation materials to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeation of the composition.

[0460] Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin). rhodextrins; fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose or dextrins); proteins (e.g., serum albumin, gelatin or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide). solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); osmolality enhancers (e.g., alkali metal halides (preferably sodium chloride, Suitable diluents include, but are not limited to, sodium thorium or potassium, mannitol sorbitol; delivery vehicle; diluent; excipient and / or pharmaceutical adjuvant. Neutral buffered saline or saline mixed with homologous serum albumin are examples of suitable diluents. Preservatives, such as benzyl alcohol, may also be added according to appropriate industry standards. The composition may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Suitable components are non-toxic to recipients at the dosages and concentrations used.

[0461] Optionally, the composition further comprises one or more additional agents, e.g., physiologically active agents, e.g., antiangiogenic agents, chemotherapeutic agents (e.g., capecitabine, 5-fluorouracil or doxorubicin), analgesic agents, etc., non-exclusive examples of which are provided herein.

[0462] In another embodiment of the present disclosure, the compositions disclosed herein can be formulated in neutral or salt form.Exemplary pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein) and salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, solutions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.

[0463] Carriers may further include any solvent, dispersion medium, vehicle, coating, diluent, antifungal and antimycotic agent, isotonic and absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to humans.

[0464] The optimal pharmaceutical composition is determined by those skilled in the art, depending on, for example, the intended route of administration, the delivery format and the desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra.Such compositions can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the polypeptide.For example, suitable compositions can be water for injection, physiological saline solution for parenteral administration.

[0465] In some embodiments, the active ingredient (e.g., ABP, ABP-drug conjugate) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, or 25 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

[0466] In some embodiments, the pharmaceutical composition further comprises, in addition to a protein or polypeptide of the present disclosure, one or more additional active ingredients, which are one or more of the additional agents described in 3.5.1.

[0467] The pharmaceutical composition may be formulated for administration by any route of administration suitable for human or veterinary medicine. In some embodiments, the pharmaceutical composition is adapted for injection. In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, intraperitoneal or subcutaneous administration. In some embodiments, the pharmaceutical composition is adapted for intravenous infusion. In some embodiments, the pharmaceutical composition is formulated for intrathecal or intraventricular administration.

[0468] In some embodiments, the dosage of the pharmaceutical composition is in the range of 100,000 cells / kg to 25 million cells / kg. In some embodiments, the dosage of the pharmaceutical composition is in the range of 100,000 cells / kg to 15 million cells / kg. In various embodiments, the dosage of the pharmaceutical composition is in the range of 100,000 cells / kg to 10 million cells / kg. In various embodiments, the dosage of the pharmaceutical composition is in the range of 100,000 cells / kg to 25 million cells / kg. In some embodiments, the dosage of the pharmaceutical composition is in the range of 300,000 cells / kg to 10 million cells / kg. In some embodiments, the dosage of the pharmaceutical composition is in the range of 300,000 cells / kg to 25 million cells / kg.

[0469] In various embodiments, the unit dosage form is a vial, an ampoule, a bottle, or a pre-filled syringe. In various embodiments, the unit dosage form is administered in an infusion bag. In some embodiments, the unit dosage form contains at least 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 125 mg, 150 mg, 175 mg, or 200 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 250 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 1×10 4 , 1×10 5 , 1×10 6 , 1.5×10 6 , 1×10 7 , 2×10 6 , 2.5×10 6 , 2.5×10 7 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5×10 7 , 1×10 8 , 2.5×10 8 , 5×10 8 , 7.5×108 , 1×10 9 , 2.5×10 9 , 5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 or 1×10 9 In some embodiments, the unit dosage form contains at least 1.5×10 immunoresponsive cells expressing a CAR. 4 , 1.5×10 5 , 1.5×10 6 , 1.5×10 7 , 2.5×10 7 , 5×10 7 , 1×10 8 , 2.5×10 8 , 5×10 8 , 7.5×10 8 , 1×10 9 , 2.5×10 9 , 5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 or 1×10 9 The subject invention relates to a method for the preparation of a CAR-expressing immunoresponsive cell.

[0470] In typical embodiments, the pharmaceutical composition of the unit dosage form is in liquid form.In various embodiments, the unit dosage form contains between 0.1mL and 50ml of the pharmaceutical composition.In some embodiments, the unit dosage form contains 1ml, 2.5ml, 5ml, 7.5ml, 10ml, 25ml or 50ml of the pharmaceutical composition.

[0471] In certain embodiments, the unit dosage form is a vial containing 1 ml of the pharmaceutical composition at a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml. In some embodiments, the unit dosage form is a vial containing 2 ml of the pharmaceutical composition at a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml.

[0472] In some embodiments, the pharmaceutical composition in unit dosage form is in a solid form, such as a lyophilizate suitable for solubilization.

[0473] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. EXAMPLES

[0474] 4. Working Example 4.1. Example 1: Identification of Tumor-Specific Antigens Tumor specific antigens (TSAs) were identified from AML patient samples as summarized in FIG.

[0475] Briefly, candidate genes for tumor-specific antigens were obtained from in silico target antigen discovery analysis by applying an improved selection process. Genes were selected for those highly expressed by AML blasts according to a gene expression profile (GSE75461) previously generated for risk stratification from 85 pediatric AML samples by the HTA 2.0 (Affymetrix) platform. The HTA 2.0 (Affymetrix) platform maximizes the collection of useful information by minimizing conserved sequences synthesized on the array. This high-resolution array design contained an unprecedented 6 million more probes covering coding and non-coding transcripts. 70% of the probes on the array covered exons of coding transcripts, and the remaining 30% of the probes on the array covered exon-exon splice junctions and non-coding transcripts. The unparalleled coverage of this array provided insight into all available coding and non-coding transcripts.

[0476] The data were used to identify highly expressed genes, i.e., genes for which all 85 AML samples had expression above the upper inflection point of the distribution (>3.4), but the healthy bone marrow control samples had no such expression (overexpressed genes by limma contrast of three HTA-arrays obtained from the AML patient cohort, and three bone marrow samples from healthy pediatric donors, after Benjamini-Hochberg multiple correction).

[0477] Among these genes, they were identified as cell surface protein antigens according to the validated surfaceome protein dataset http: / / wlab.ethz.ch / cspa / . A subset was selected if the expression was consistent with the CSPA (Computer Sensitive Areas and Polymorphisms). Candidates from this process were then validated using a public dataset, the TARGET database, from which gene expression matrices of 262 AML pediatric samples were downloaded. The TARGET data were RMA-normalized; the 85 patient cohort of Italian AML and the 262 TAGET AML samples were z-score normalized, which allowed the comparison of the relative expression of selected genes via box plots between the two cohorts. This allowed the identification of 44 genes (first list - FL) not expressed in healthy donor bone marrow as the best representative surface antigens of pediatric AML at the time of disease onset.

[0478] This first list - 44 candidate genes in FL were then analyzed by querying public databases for broad coverage and specificity, with the aim of selecting the most promising targets to enter the following in vitro testing of protein expression. GeneCards software was used to explore the functional description of TSAs, gene chromosomal localization, association with known pathologies / cancers, mRNA expression in normal human tissues, predicted protein expression, subcellular location, and involved pathways; Pubmed was used to obtain information about previous involvement of TSAs in cancers, including AML, and to exclude their involvement in previous CAR-T development projects; and XenaBrowser was used to predict the expression selectivity of candidate TSAs in AML. It is an online platform for exploring public and private, multi-omic and clinical / phenotypic data sets, including 1500+ datasets and 50+ cancer types. The tool provides interactive online visualization of influential cancer genomics datasets, including data from The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), Genomic Data Commons (GDC) and the UCSC RNA-seq compendium. Xena supports virtually any functional genomic data, including SNVs, INDELs, large structural variants, copy number variations, genes, transcripts, exons, miRNAs, lncRNAs, protein expression, DNA methylation, ATAC-seq signals, phenotypic annotations and higher-level derived genomic parameters.This process of refinement used gene function (associated with cancer) and localization on the cell surface as inclusion criteria, and the following exclusion criteria: i) function considering enzyme and ribosomal components, ii) localization other than the cell surface, including the nuclear membrane; iii) previous involvement in AML diagnostic panels or CAR-T development, iv) having previously been part of a patent as judged by Espacenet, Trade Mark information, patentscope, and v) reported high expression in multiple human tissues. At the end of this refinement process, 26 of the 44 genes in FL were discarded, providing 18 candidate TSAs.

[0479] Applicants then generated a second list - SL, by querying the same in silico dataset and selecting genes whose expression was below the inflection point of the distribution (>2.4 and <3.4). This new list contained an additional 32 candidate TSAs that underwent the same analytical process described above for TSA refinement. Twenty-four of the 32 TSAs identified in SL were discarded due to exclusion criteria (i-v), providing eight additional candidate TSAs.

[0480] These 26 candidate TSAs (18 candidate TSAs from FL and 8 additional candidate TSAs from SL) were then characterized for protein expression and localization by flow cytometry (FC). Commercially available primary antibodies recognizing the candidate TSAs were first tested with positive control cells. Once the antibodies were demonstrated to provide staining above control, cell surface staining for the TSAs was performed on a minimum of two AML cell lines (SHI-1 and HL-60). Eleven of the 26 candidate TSAs were confirmed to be expressed on the AML cell surface by flow cytometry (either strongly or weakly expressed, as shown in Figure 1).

[0481] Six AML-TSAs (CD63, CD151, CD72, CD84, CD69, and CD109) were finally selected based on their strong positive signals and specificity detected by flow cytometry using AML cell lines SHI-1 and HL-60 (Figure 1). For further validation, their expression on additional AML cell lines, including KASUMI-1, MOLM-13, and MV4-11, was examined by immunofluorescence using the same antibodies used for flow cytometry.

[0482] Flow cytometry and fluorescent immunohistochemistry data are provided in Figures 2A and 2B (CD69), 3A and 3B (CD63), 4A and 4B (CD151), 5A and 5B (CD84), 6A and 6B (CD109), and 7A and 7B (CD72). As provided in Figures 2C (CD69), 3C (CD63), 4C (CD151), 5C (CD84), 6C (CD109), and 7C (CD72), antigen expression was specific to AML cells - much lower or no expression was detected by flow cytometry on fractionated subpopulations obtained from healthy donor peripheral blood mononuclear cells (PBMCs) including healthy hematopoietic cells: CD19+ B lymphocytes, CD3+ T lymphocytes, CD33+ myeloid precursor cells, and on CD34+ cells extracted from umbilical cord blood samples. CD34 in healthy BM subpopulations in normal regenerating bone marrow + and CD34 + CD38 - Expression of TSA in the gated blasts of acute lymphoblastic leukemia patients, in the gated blasts of AML patients at diagnosis, in the gated blasts of AML patients at relapse, in the gated blasts of AML patients with residual blasts, as well as in the gated blasts of patients with other malignancies, e.g., myeloid neoplasms, in subpopulations, are also summarized in Tables 1-8 below, respectively.

[0483] Table 1: Expression of selected TSAs in healthy blood mononuclear cell subpopulations. Table 1 provides the expression of TSAs in healthy blood samples and showed heterogeneous dim expression of TSAs in peripheral blood mononuclear cell subpopulations. [Table 1] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification) Table 2: Expression of selected TSA subpopulations in normal regenerating bone marrow. [Table 2] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0484] Table 2 provides TSA expression in healthy subpopulations from regenerating bone marrow collected at the end of treatment, showing low / dim expression in the myeloid lineage subpopulation in 3 / 6 TSAs. Table 3: CD34 during normal regeneration + and CD34 + CD38 - Expression of selected TSAs in subpopulations [Table 3] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0485] Table 3 shows regenerating bone marrow-derived stem-progenitor CD34 cells collected at the end of treatment. + CD38 - TSA expression in subpopulations was provided, showing weak expression for some of the TSAs. Table 4: Expression of TSA in B or T acute lymphoblastic leukemia at diagnosis [Table 4] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0486] Table 4 provides TSA expression in acute lymphoblastic leukemia samples at diagnosis and shows all TSAs appropriate for detecting B lymphoblasts, whereas T lymphoblasts exclusively expressed CD69 and CD84. Table 5: Expression of TSA in AML at diagnosis [Table 5] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0487] Table 5 provides TSA expression in acute myeloid leukemia samples at diagnosis, showing all TSAs appropriate for detecting myeloid blasts, with CD69 and CD84 being the most specific. Table 6: Expression of TSAs in AML at relapse [Table 6] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0488] Table 6 provides TSA expression in acute myeloid leukemia samples at diagnosis, showing all TSAs suitable for detecting blasts at disease flares, with CD69 and CD84 being more specific. Table 7: Expression of TSA in treated AML with residual blasts (<5%) [Table 7] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0489] Table 7 provides TSA expression in bone marrow samples collected during treatment with myeloid residual disease ranging between 0% and 5% blasts, with CD69 and CD84 being most appropriate for detecting low disease levels. Table 8: Expression of TSA in myeloid neoplasms [Table 8] Strong positive (WHO classification) Weak positive (WHO classification) POS B: Positive bright POS D: Positive dim POS M:Positive Intermediate PP1:Partially positive 1 POS H: Positive heterogeneous (AIEOP-BFM classification) PP2: Partial positive 2 (AIEOP-BFM classification)

[0490] Table 8 provides TSA expression in bone marrow samples of myeloid neoplasms at the time of diagnosis and demonstrated the ability of CD69, CD84 and CD72 to reveal dysplastic cells.

[0491] Specific expression was further confirmed by observing no or low expression of TSA on healthy donor primary fibroblasts (Figure 8).

[0492] Expression of the TSAs was also examined in other cancer cell lines representing solid and liquid tumors distinct from AML (Figure 10). The six TSAs were expressed at various levels, but overall expression levels were low in these other cancer cells.

[0493] These studies show that the six selected TSAs are robustly and selectively expressed in the majority, if not all, AML cell lines tested. High expression of the six TSAs was also evaluated and confirmed using two AML patient-derived explants (PDXs) (generated from primary pediatric AML samples) available in the laboratory by examining their gene expression profiles.

[0494] The stability and predictive value of TSAs were investigated by evaluating their mRNA expression in pediatric AML samples obtained for diagnosis or to test for remission after treatment according to genetic risk stratification. High expression of TSAs was identified during the active phase of disease (at diagnosis) and reduced expression was identified in samples collected at the end of treatment. method

[0495] Patient samples and cell lines. Bone marrow (BM) samples were collected, processed and analyzed according to standardized operating procedures. 42 samples from patients with AML de novo - 30 at diagnosis and 12 at relapse. 4 samples from myeloid neoplasms, 7 samples of AML collected during treatment with residual blasts (<5%), 6 samples of B-ALL at diagnosis and 1 sample of T-ALL, as well as 13 BM samples collected during follow-up when in disease remission, characterized as regenerating, as previously defined (Buldini B. et al., BJH 2018).

[0496] All AML cell lines were purchased by DSMZ. HL-60, Kasumi-1 and MOLM-13 were cultured in RPMI (GIBCO, Life Technologies, Paisley, UK) supplemented with 1% penicillin-streptomycin (GIBCO, Life Technologies, Grand Island, NY, USA), 1% L-glutamine (GIBCO) and 10% fetal bovine serum (GIBCO). MV4-11 and SHI-1 were cultured in Dulbecco's modified medium (GIBCO) supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% fetal bovine serum.

[0497] Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of healthy donors with informed consent by Ficoll density separation. CD34 positive cells were obtained from umbilical cord blood of healthy donors with informed consent by Ficoll density separation followed by magnetic isolation on columns from the CD34 MicroBead Kit (Miltenyi Biotech GmbH, Bergish Gladbach Germany) according to the protocol. Primary cells from whole blood (maximum 100 μL per tube) were stained with conjugated antibodies. Flow cytometry analysis.

[0498] BM samples were stained with primary conjugated antibodies for 15 min in the dark, then lysed and centrifuged. Cells were resuspended in PBS and analyzed by BD FACS CANTO II acquiring a minimum of 30,000 events.

[0499] The primary antibodies used for the clinical multiparameter analysis (Table 4) were the following anti-human antibodies: CD45 V500 (BD Pharmingen, Cat. No. 560777), CD45 APC-Cy7 (BD Pharmingen, Cat. No. 348815), CD34 PC5 (Beckman Coulter, Cat. No. A07777), CD38 PE-Cy7 (BD Pharmingen, Cat. No. 335825), CD33 APC (BD Pharmingen, Cat. No. 551378), CD7 V450 (BD Pharmingen, Cat. No. 642916), HLA-DR V500 (BD Horizon, Cat. No. 561224), CD33 PC5 (Beckman Coulter, Cat. No. I112647), CD117 PC7 (Beckman Coulter, Cat. No. IM3698), CD34 A750 (Beckman CD69 A488 (R&D Systems, catalog number FAB23591G), CD63 PE (BD Pharmingen, catalog number 556020), CD151 PE (R&D Systems, catalog number FAB1884P), CD84 APC (Biolegend, catalog number 326010), CD109 A488 (R&D Systems, catalog number FAB23591G), CD38 V450 (BD Pharmingen, catalog number 561378), CD19 PE (exbio, catalog number 1P-305-T100), CD7 PC5 (Beckman Coulter, catalog number I113613), CD34 APC (BD Pharmingen, catalog number 345804), CD3 APC-Cy7 (BD Pharmingen, catalog number 341110). Systems, catalog no. FAB4385G), and CD72 BV421 (BD Pharmingen, catalog no. 743794). [Table 9]

[0500] Table 9 shows an antibody diagnostic panel for immunophenotyping at the time of diagnosis by flow cytometry of acute leukemia or myeloid neoplasms. Intracellular staining for antibody validation.

[0501] All primary antibodies listed above were first validated by performing intracellular staining of PBMCs as suggested by the guidelines.

[0502] Briefly, cells (6 × 10 5 ) were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and then rinsed three times with PBS. The cells were then permeabilized with 0.1% TWEEN® 20 in PBS for 20 min and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min. The permeabilized cells were stained with primary antibodies together with Fc Receptor Blocking (Miltenyi) for 30 min and then incubated with secondary antibodies for 20 min. Cell surface staining.

[0503] Primary antibodies were tested for their binding on the surface of CD34 positive cells derived from AML and other cancer cell lines, PBMCs, and umbilical cord blood. Cells (3×10 5 100 pieces) were blocked with 3% BSA in PBS and stained with primary antibodies for 20 min with Fc Receptor Blocking. After washing with PBS, the cells were stained with secondary antibodies for 20 min.

[0504] For PBMC subpopulations, antibodies against TSA were used for 20 min together with the following anti-human antibodies: CD3-APC (Beckam Coulter, Marseille, France), CD19-PE (Beckam Coulter), CD33-PC5 (Beckam Coulter). Cells were then stained with secondary antibodies for 20 min. Cells were analyzed using CytoFLEX (Beckam Coulter) and Flow Jo software (version 9.7, TreeStar Inc.). Immunofluorescence.

[0505] Primary antibodies used in these analyses were from eBioscience, Abcam and BD; secondary antibody was goat anti-mouse IgG Alexa488 antibody (Life Technologies). Cells (3 × 10 5 ) were seeded on the bottom of culture chamber slides (FALCON, Big Flats, NY, USA) pre-coated with fibronectin (40ug / ml) (Corning) for 2 hours at 37°C. After this incubation period, cells were stained with MemBrite Fix dye (Biotium), a specific membrane dye, for 5 minutes at 37°C according to the guidelines. Cells were fixed with 4% formaldehyde in PBS for 15 minutes, washed, and then blocked with 3% BSA in PBS for 30 minutes. After saturation, cells were stained with primary antibodies together with Fc Receptor Blocking overnight at 4°C, and then with secondary antibodies for 1 hour at room temperature. Cells were imaged with a Zeiss 2.6 laser scanning microscope. Images were analyzed using Image J win 32 software. 4.2. Example 2: Methods of Treatment Targeting AML-TSA

[0506] Six AML-TSAs (i.e., CD63, CD151, CD72, CD84, CD69 and CD109) were tested as described in Example 1 and selected based on i) robust broad expression in primary myeloid neoplasms, acute leukemias and acute myeloid leukemias, ii) lack of expression / low expression in CD34+CD38+ HSPCs, iii) association with tumor development, and iv) expression in AML cell lines.

[0507] For the development of a therapy targeting one of the TSAs, two to four different commercially available antibodies against each target were tested to design the best single-chain variable fragment (ScFv) against each TSA. For each of the antibodies, flow cytometry results against AML cell lines are provided in Figure 8.

[0508] Additionally, novel antibodies against TSA are generated using methods known in the art, for example, procedures involving immunogen preparation, immunization, hybridoma production, screening and purification, or procedures for panning phage-displayed naive or immunized antibody libraries. The novel antibodies are also tested for their binding affinity and specificity for the TSA target.

[0509] The antibody selected for its binding affinity and specificity is used for the development of ABP-drug conjugate and CAR-T cell. The ABP-drug conjugate is generated by conjugating the selected antibody to a cytotoxic agent. The CAR is generated by using the antigen-binding domain of the selected antibody as an extracellular domain. The polynucleotide encoding the CAR is generated, which includes an antigen-binding domain, a transmembrane domain, a signal transduction domain, and optionally at least one co-stimulatory domain. The polynucleotide is transfected into T cell or NK cell to generate CAR-T cell or CAR-NK cell.

[0510] Each of the ABPs, ABP-drug conjugates and CAR-T cells is tested in vitro using AML cell lines and primary pediatric AML samples.When the ABPs, ABP-drug conjugates or CAR-T cells are applied to the cell culture of AML cell lines or primary AML cells, they induce cytotoxic effects specifically against cancer cells.

[0511] The ABP-drug conjugate and CAR-T cells are also tested in vivo using NSG (NOD / scid IL-2Rgnull) mice (syngeneic human disease model) intravenously injected with AML cell lines, and by using patient-derived xenograft (PDX) animal models. Cancer cells in the animal models are reduced in peripheral blood, bone marrow or spleen after administration of ABP, ABP-drug conjugate or CAR-T cells. 4.3. Example 3: Methods of Treatment Targeting AML-TSA Using CAR-T Cells

[0512] Validation of the use of selected TSAs (CD63, CD151, CD72, CD84, CD69 and CD109) as targets for T cell-immunotherapeutic approaches based on the use of the generated CAR constructs.

[0513] Six tumor-associated antigens, namely CD69, CD63, CD151, CD84, CD109 and CD72, were identified as uniquely associated with childhood acute leukemia and myeloid disorders. CD69 and CD84 antigens were characterized in vitro and in vivo and demonstrated to be highly specific for primary acute myeloid leukemia (AML) samples collected at diagnosis and at relapse. CD69 and CD84 antigens were found to be highly expressed in myeloid disorders, including acute myeloid leukemia (AML) bone marrow samples collected at diagnosis (in 75% and 96% of cases, respectively) or at relapse, and in samples collected after treatment, samples with residual disease defined as >1 blasts and ≦10%. Interestingly, CD72 was expressed in the majority of B-cell precursor acute lymphoblastic leukemia samples examined at diagnosis (98% of BCP-ALL), as well as in the majority of AML cases at diagnosis (66%).

[0514] Based on this data, eleven novel single chain variable fragment (ScFv) sequences (A1, C1, F1, G1, C2, F2, H2, H3, F12 and B8) were developed by phage display to generate chimeric antigen receptors (CARs) that recognize CD69 or CD84, or a combination of CD69 and CD84, on AML blasts. The CD72 antigen was shown to be highly specific for acute lymphoblastic leukemia (ALL) and had great potential for the diagnosis of ALL patients treated with CD19-directed immunotherapy, who frequently lose the CD19 marker. Design and manufacture of novel CD84 and CD69 CAR-T cells

[0515] Two novel antigens, CD84 and CD69, were prioritized as potential targets for AML immunotherapy. Phage display was used to find high affinity antigen binders for both antigens from a large combinatorial library containing up to billions of antibody targets. The method considered six rounds of panning, by enriching the population of phages with high affinity, exposing the library to our antigen, and then eluting and amplifying only those with the highest binding affinity, as an iterative process to enrich for phages in the phage population with high affinity binding to our target compared to others. This qualitative selection process identified 87 clones for CD69 and 6 clones for CD84. Validation using an ELISA technique that allows for more accurate evaluation of antibody-antigen interactions by quantitative immunoassay confirmed eight and two ScFv sequences that were good candidates for CD69 and CD84, respectively. By gene synthesis, 10 novel ScFv genes were produced, which were then cloned into a third-generation chimeric antigen construct (CAR) containing CD28 H / TM and 4-1BB costimulatory domains in combination with the zeta (CD3ζ) signaling domain in a pUC57 plasmid. By restriction enzyme technology, the CAR was excised and pasted into a third-generation lentiviral transfer plasmid for high-titer lentiviral vector (LV) production (Figure 13). The workflow then included isolation of healthy donor T cells, followed by efficient activation, gene transfer of the CAR construct into the activated T cells, CAR-T cell expansion, phenotyping and analysis of the final CAR-T cell product after co-culture alone (---) or with the target AML cell line.

[0516] CAR-T cells were produced using four different specific ScFvs identified within a human naive antigen-binding fragment (Fab) phage library, namely 14722-P1-F12 and 14722-P1-B8, which recognize CD84, and 14721-P1-H3 and 14721-P1-G1, which recognize CD69. The nucleotide (SEQ ID NOs: 24-34) and amino acid sequences (SEQ ID NOs: 35-44) of the four CAR constructs are provided in FIG. 27. Prior to viral transduction, T cells were isolated from peripheral blood mononuclear cells (PBMCs) collected from healthy donors by magnetic enrichment using CD4 and CD8 MicroBeads and subsequently activated in T Cell TransAct™ medium. Then, after 48 hours of expansion in TexMACS™, T cells were transduced with LVs encoding the CAR at a multiplicity of infection (MOI) of 10 in IL-7 and IL-15 supplemented medium. After 24 hours of transduction, the vector was washed out and the CAR-T cells were subjected to 14 days of expansion (Figure 14). Transduction efficiency was measured by digital droplet polymerase chain reaction (ddPCR) and expressed as vector copy number (VCN) per cell; the average VCN ranged between 2.5 and 33.7 in all CAR-T cells produced, and there were no significant differences in VCN between different CAR-T products (Figure 15). Mock-transduced T cells (empty CAR) were used as a control.

[0517] The potency and specificity of newly produced CD84 and CD69 CAR-T cells were evaluated in To be properly tested in vitro and in vivo, AML cell lines were first screened for cell membrane expression of CD84 and CD69 by flow cytometry. HL-60 and SHI-1 expressed high levels of both antigens and were target cells (Figure 16); MV4-11 and MOLM-13 expressed only CD84, whereas Kasumi-1, U937 and K562 cells expressed only CD69 (Figure 16) and were suitable as control cell lines for CD69 or CD84, respectively.

[0518] The CAR design does not affect the expansion, viability and phenotype of CD84 and CD69 CAR-T cells.

[0519] Enumeration of manufactured CAR-T cells at the end of 14 days of expansion (corresponding to day 17 of the CAR-T cell manufacturing process) showed expansion rates ranging between 2-fold and 20-fold (FIG. 17), with high variability due primarily to the starting donor PBMCs (FIG. 18A). T cells were viable and CD8 + CD4 rather than cells + Cells were enriched (Figure 18B), the majority of which, as expected, were naïve and stem cell memory (T n +T scm , CD197[CCR7] + CD45R0 - ), and Central Memory (T cm , CD197(CCR7) + CD45R0 + ) T cells (Figures 19A and 19B). Upon expansion, CD84 and CD69 CAR-T cells were both activated and exhausted to the same extent as empty CAR-T cells, as indicated by upregulation of CD25, CD154, CD107a, and CD137, as well as upregulation of CD366, CD223, and CD279, in response to culture conditions. It is noteworthy that the same results were observed with control T cells (empty CAR), confirming that the CAR cassette did not alter T cell function (Figures 20A and 20B). CD84 and CD69 CAR-T cells have potent anti-tumor lytic activity in vitro

[0520] To test the activity of the newly generated CD84 and CD69 CAR-T cells, the CAR-T cells were maintained in medium alone (---) or in co-culture with the target AML cell lines expressing the antigen. Significant in vitro antitumor activity was measured with the four ScFv sequences tested against the target cell lines, ranging between 10% and 80% against HL-60 and between 50% and 90% against SHI-2 cells. These results are summarized as follows: 1) CD33+ Frequency of dead cells (Annexin V + and / or 7AAD + , FIG. 21), 2) lytic efficacy (FIG. 22), and 3) bioluminescence signal in luciferase-transduced AML cells (FIG. 23). The persistence of activated CAR-T cells was also monitored in vitro for up to 48 hours by cell counting. CAR-T cell activity is characterized by increased proliferation of T lymphocytes (black squares) with a concomitant reduction in the number of target AML cells (white squares) (FIG. 24).

[0521] Specific lytic activity of AML target cell lines with A1, F1, C2 and H2 ScFv(s) targeting target cell lines expressing CD69 was demonstrated in vitro.Specific lytic activity of primary AML cells derived from pediatric AML xenografts (PDX) by CAR-T with anti-CD-84 B8 and F12 ScFv(s) was demonstrated in vitro.

[0522] CD84 and CD69 CAR-T cells express CD34 + No toxicity to hematopoietic stem and progenitor cells (HSPCs)

[0523] CD34 + The potential off-target activity of CD84 and CD69 CAR-T cells against hematopoietic stem and progenitor cells (HSPCs) was examined by standard colony-forming unit (CFU) assays at an effector:target ratio (E:T) of 1:1. Both CD84 and CD69 CAR-T constructs expressed CD34 + The IL-16 expression level did not produce any significant effect on CD34 cells, suggesting that these + A significant reduction in the number of CFUs formed by HSPCs was also observed in CD34 + T cells expressing CD84 and CD69 CARs recognize and kill AML cell targets in vivo.

[0524] To test CAR-T cell activity in vivo, CAR-T cells targeting CD84 and CD69 were generated and injected into NOD / SCID gamma (NSG) mice engrafted with targeted AML cell lines. The AML cell lines were engineered to express luciferase for non-invasive bioluminescence imaging (BLI). Briefly, mice were injected with 0.5×10 6 AML cells / mouse were injected via tail vein (day 1), followed by (day 3) 1.5 × 10 6 Mice were injected with CAR-T cells or mock-transduced T cells (empty CAR at MOI 10 / mouse; n=6-8 per condition tested). Mice treated with B8 and F12 (CD84) and G1 and H3 (CD69) CAR-T cells experienced a statistically significant reduction in leukemic burden as assessed by BLI already at 14 days after AML challenge, which became even more evident over time (at days 23 and 35) (Figures 26A, 26B, 26C). material and method

[0525] Human cell lines

[0526] HL-60 and SHI-1 AML cell lines were used as target cells for TSA screening. These cell lines were grown in RPMI (Life Technologies; 11875093) supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106), 1% penicillin-streptomycin (P / S, 10000U / mL, Life Technologies; 15140148) and 1% L-glutamine (200mM, ThermoFisher; 25030024), except for the SHI-1 cell line, which was cultured in DMEM (Life Technologies; 41965039) supplemented with 10% FBS and 1% P / S. HEK293T were grown in IMDM (Euroclone; ECB2072L) supplemented with 10% FBS, 1% P / S and 1% L-glutamine. All cells were incubated at 37 °C for 24 h at 20 °C for 30 min at 37 °C for 1 h at 20 °C. 2 The cells were cultured at 37°C in a humidified incubator containing

[0527] Cloning

[0528] The CAR cassette (1506 bp) was cloned from the pUC57 vector (synthesized by ProteoGenix SA) into the third generation LV transfer plasmid under the control of the human phosphoglycerate kinase promoter (hPGK). One Shot TOP10 chemically competent E. coli (ThermoFisher, Walthan, MA, USA) was transformed and DNA was extracted using the Plasmid DNA Maxiprep Kit (Thermo Fisher Scientific; K210017). The procedure was controlled by digestion with BamHI and SalI enzymes and agarose gel electrophoresis. Sanger sequencing of the plasmid was performed to verify the correct insertion of the cassette in the backbone.

[0529] CD4 from PBMCs + and CD8 + Isolation and T cell expansion

[0530] PBMCs were isolated from healthy donors by density gradient centrifugation using Lymphoprep (StemCell technologies; 07861). + and CD8 +T cells were magnetically isolated using an autoMACS instrument (Miltenyi Biotec; Bergisch Gladsbach, Germany) with CD4 and CD8 MicroBeads (Miltenyi Biotec; 130-045-101 and 130-045-201, respectively) according to the manufacturer's instructions. Isolated T cells were then activated on day 1 for in vitro expansion using TransAct (Miltenyi Biotec; 130-111-160) in TexMACS medium (Miltenyi Biotec; 130-097-196) supplemented with recombinant human IL-7 (500 IU / ml) and IL-15 (84 IU / ml) (Miltenyi Biotec; 130-095-362 and 130-095-764, respectively) containing 1% P / S. On day 4, cells were washed from TransAct and lentivirus and then resuspended at 1-2 x 10 cells per mL. 6 Cells were maintained in TexMACS containing IL-7 and IL-15.

[0531] Lentiviral vector production and transduction of human T cells

[0532] LV was produced via transient transfection of the HEK293T packaging cell line as previously described (Langford-Smith et al., 2012). Briefly, 70% confluent cells were co-transfected with Gag / Pol(III gen), Env and Rev packaging plasmids, pAdvantage plasmid (pADV), and transfer vector plasmid carrying the cassette. 48 hours after transfection, lentiviral supernatants were collected, ultracentrifuged, aliquoted, and stored at -65°C or below.

[0533] T cells were transduced with LV at 10 MIO with 0.01 mg / mL Vectofusin-1 (Miltenyi Biotec; 130-111-163) on day 3. 16 hours after transduction, cells were washed from LV and TransACT. To evaluate transduction efficiency, VCN per cell was measured. Total genomic DNA from transduced T cells was extracted 14 days after transduction using Dneasy Blood & Tissue Kit (Qiagen; 69504). To determine VCN per cell, ddPCR was used with a reaction mixture containing ddPCR Supermix for Probes without dUTP (Bio-Rad; 1863024) and primer-probe sets for target and reference genomes (ddPCRTM CNV Assay [FAM] 10031277; ddPCRTM CNV Assay [HEX] 10031244). Droplets were generated with an Automated Droplet Generator (Bio-Rad) and read with a QX200 droplet reader (Bio-Rad). VCN was analyzed using QuantaSoft droplet reader software and determined by the ratio of the target gene concentration to the reference gene concentration, multiplied by the copy number of the reference gene in the reference genome.

[0534] Activation, exhaustion and differentiation markers by flow cytometry

[0535] Cells were stained with fluorochrome-conjugated primary antibodies and isotype controls for 15 minutes at room temperature. Stained cells were washed and immediately analyzed using a FACSCelesta™ Cell Analyzer (BD Biosciences). The following antibodies were used: CD3 (PE-Vio615, Miltenyi Biotec; 130-114-520), CD4 (VioGreen, Miltenyi Biotec; 130-113-230), CD8 (APC, Miltenyi Biotec; 130-110-681), CD45 (VioBlue, Miltenyi Biotec;130-110-637), CD34(PE, Miltenyi Biotec;130-124-456), CD38(APC, Miltenyi Biotec;130-123-852), CD197(CCR7)(VioBlue, Miltenyi Biotec;130-117-353), CD45RO(APC, Miltenyi Biotec;130-113-556), CD223(VioBlue, Miltenyi Biotec;130-118-549), CD279(PE, Miltenyi Biotec;130-120-385), CD366(APC, Miltenyi Biotec;130-119-781), CD154(VioBlue, Miltenyi Biotec;130-116-615), CD25(PE, Miltenyi Biotec;130-114-541), CD137(APC, Miltenyi Biotec;130-110-764), CD69(PE, Miltenyi Biotec; 130-112-613), CD84 (APC, Biolegend; 326010), CD107a (PE, Miltenyi Biotec; 130-111-621) and CD33 (FITC, Miltenyi Biotec; 130-111-018). Analysis was performed by using FlowJo software (BD Biosciences, v10).

[0536] In vitro cytotoxicity assay

[0537] Cytotoxicity assays were performed at the end of T cell expansion (day 17) by co-culturing AML target cell lines (positive for antigen) with CAR-T cells or non-transduced T cells for 48 hours at an E:T ratio of 1:1. Co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry with a FACSCelesta™ Cell Analyzer and FlowJo software. The percentage of cell lysis was calculated using the following formula:

[0538]

number

[0539] Alternatively, the bioluminescence of luciferase-transduced AML cells was analyzed 48 hours after co-culture with CAR-T cells. Briefly, cells were centrifuged, resuspended in 50 μl of PBS 1×, and then incubated with XenoLight D-luciferin firefly (15 mg / mL in PBS; Perkin Elmer, Waltham, MA) for 10 minutes. Luciferase activity was measured by a Spark-Tecan multi-well plate reader (Tecan Group Ltd., Mannedorf, Switzerland), and data signal reduction is reported as the percentage of AML cell lysis assessed by bioluminescence imaging (BLI). Colony formation assay

[0540] After 6 h of coculture at a 1:1 ratio, a total of 2 × 10 3 The first CD34 +Cells (isolated from healthy bone marrow or umbilical cord blood) were seeded in 500 μl of MethoCult™ (H4534, Stemcell Technologies, Meda MB, Italy) in 24-well plates and incubated at 37° C. Four replicates were plated for each co-culture. 14 days after seeding, an appropriate volume of a 1:6 solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich-Merck) in Hanks was added to the semi-solid medium. Images were acquired by a light microscope equipped with a camera and colonies were counted using ImageJ software. In vivo experiments in NSG mice

[0541] Procedures involving animals and their care were carried out in accordance with national and international laws and policies (EEC Council Directive 86 / 609, OJ L 358, 12 December 1987) as well as institutional guidelines adhering to the "ARRIVE" guidelines (Animals in Research Reporting In Vivo Experiments). Ministerial Authorisation Approval: 131 / 2022-PR. NSG mice (NOD.Cg-PrkdcscidII2rgtm1Wjl / SzJ, 4-5 week old female, 20-25 g / mouse, max 5 animals / cage) were incubated with 0.75 × 10 6 SHI-1-LUC (transduced with luciferase gene) cells were injected intravenously (tail vein). Two days after AML injection, mice were transfected with 1.5 × 10 6 Tumors were treated by intravenous injection of CAR-T or non-transduced / mock-transduced T cells as a control. Bioluminescence was monitored by intraperitoneal injection with XenoLight D-luciferin firefly (15 mg / ml in PBS; Perkin Elmer) 10 min prior to measurement (Xenogen IVIS Spectrum Bioluminescence / Optical Imaging System, Xenogen Corporation, Alameda, Calif.) to verify tumor growth.

[0542] statistical analysis

[0543] Significant differences in means were tested by either Student's t-test or Wilcoxon nonparametric test according to the distribution of means. When comparing more than two groups / conditions, one-way multiple comparison ANOVA was used with Tukey's multiple comparison test. Graphs and related statistical analyses were generated using GraphPad Prism 8. All data are presented as mean ± standard error of the mean (SEM). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** A value of P<0.0001 is statistically significant. result

[0544] The data presented herein confirms that:

[0545] High expression of all six antigens in multiple primary cases collected at diagnosis and at relapse of myeloid and lymphoid acute leukemia, associated with very low / null expression on healthy regenerating bone marrow and hematopoietic stem cells; and

[0546] Specific lytic activity of AML blasts by CAR-T cells created with novel ScFv(s) recognizing both CD69 and CD84 in in vitro experiments and in mouse xenografts engrafted with AML cell lines.

[0547] The data support the development of immunotherapeutic and diagnostic approaches based on CD63, CD151, CD72, CD84, CD69 or CD109 for myeloid and lymphoid leukemias. 4.4. Example 4: Validation of CAR-T Binding and Blast Lysis in Zebrafish

[0548] The efficacy of scFv binding to TSA is tested. GFP+ AML cell lines expressing high levels of TSA (as well as GFP+ cell lines not expressing TSA as a control) are injected into zebrafish embryos 48 hours after fertilization in a mixed solution with mCherry+ CAR-T or control T cells at a 1:1 ratio (hereinafter referred to as "mixed cells").

[0549] For transplanted animals, the number of circulating fluorescent GFP / mCherry positive cells and yellow-merged complexes showing binding complexes at 1, 5, 24 and 72 hours post-injection will be assessed by fluorescence microscopy and ImageJ software in all cohorts of animals (approximately 30 embryos each) to determine binding between AML and CAR T cells.

[0550] Next, scFv specificity and killing efficacy will be evaluated. Proliferation / death of xenografted CAR-T cells and reduction of AML blasts in embryos will be evaluated.

[0551] Twenty-five embryos per group are sacrificed at 5 and 24 hours post-injection to count GFP+ and mCherry+ cells by flow cytometry. Comparison between reporter-expressing specific TSA-CAR T cells and control vector indicates the lytic efficacy that occurs after binding. 4.5. Example 5: In vitro lytic potency of anti-CD84 (B8 and F12) and anti-CD69 (A1, F1, C2, and H2) CAR-T cells against primary AML cells

[0552] To test the activity of anti-CD84 CAR-T cells, CAR-T cells containing scFv B8 and F12 were cultured in medium alone (---) or in co-culture with AML cells derived from an AML patient-derived xenograft model (AML-PDX). AML-PDX generation

[0553] AML patient-derived xenografts (PDXs) were generated using blasts derived from bone marrow of pediatric patients at the time of de novo AML diagnosis. Figure 29 shows the expression of TSA CD84, CD69 and CD72 in primary AML cells derived from AML-PDXs. Specific CAR candidates from previous experiments were tested in AML-PDXs.

[0554] For PDX generation, NOD.Cg-Prkdc scid Il2rg tm1Wjl NSG / SzJ mice (4–8 weeks old) were conditioned by irradiation with 1.5 Gy 24 h prior to leukemia cell transplantation. Briefly, CD3 IgG was isolated by immunomagnetic cell separation (Miltenyi Biotec) using a CD3 MicroBead Kit (Miltenyi Biotec) to avoid graft-versus-host disease (GvHD). + T cells were prophylactically depleted at 1 × 10 6 Primary AML cells were injected intravenously (iv) into first mouse recipients (P0). To monitor engraftment, tumor challenge was monitored by measuring human (h)CD45+ / - ... + The results were evaluated by flow cytometry of cells. hCD45 in PB + Mice were sacrificed when hCD45+ cells >20% indicated AML engraftment. Organs (femurs and spleens) were harvested, flushed, and mechanically dissociated to obtain cells for biobanking. Over two consecutive passages, 1×10 6 hCD45 + The cells were injected intravenously into second and third recipient mice to generate P1 and P2-PDX models. The stability of the P2 model was tested by RNA and exome sequencing. Ex vivo cells were used for in vitro studies.

[0555] When anti-CD84 CAR-T cells, B8 and F12, were co-cultured with AML cells derived from AML-PDX models, anti-CD84 CAR-T cells B8 and F12 exhibited lytic potency against target cells, as shown in Figure 30 as "% of killing". The lytic potency of CAR-T cells B8 and F12 was observed when normalized to that of empty CAR T cells, thereby confirming that the CAR cassette did not alter T cell function and that CD84 is a specific target of primary AML. Consistently, the in vitro antitumor activity of B8 and F12 anti-CD84 CAR-T cells ranged between 5-80% in AML cells from five different AML models. The persistence of activated CAR-T cells was monitored in vitro for up to 48 hours (Figure 30).

[0556] To test the activity of the newly generated CD69 CAR-T cells, A1, F1, C2 and H2, ScFv(s) against CD69 were tested in different CAR-T cell products. CAR-T cells were maintained in culture alone or in co-culture with target AML cell lines expressing the CD69 antigen (SHI-1 and HL60 cell lines). HL-60 and SHI-1 AML cell lines were used as target cells, and U937 and K562 cell lines were used as negative controls. These cell lines were grown in RPMI (Life Technologies; 11875093) supplemented with 10% FBS, 1% P / S and 1% L-glutamine (200 mM, ThermoFisher; 25030024), except for the SHI-1 cell line, which was cultured in DMEM (Life Technologies; 41965039) supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106) and 1% penicillin-streptomycin (P / S, 10000 U / mL, Life Technologies; 15140148). HEK-293T cells were grown in IMDM (Euroclone; ECB2072L) supplemented with 10% FBS, 1% P / S and 1% L-glutamine. All cells were cultured at 37°C in a humidified incubator containing 5% CO2. Primary AML ex vivo cells were cultured at 37° C. in RPMI Medium 1640 containing 10% FBS, 2 mM glutamine (Gibco, Life Technologies), 100 U / mL streptomycin / penicillin (Gibco, Life Technologies) and supplemented with 50 ng / mL thrombopoietin (TPO), 50 ng / mL stem cell factor (SCF), 50 ng / mL FMS-like tyrosine kinase 3 ligand (Flt3L), 20 ng / mL interleukin (IL)-3 and 20 ng / mL IL-6 (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

[0557] When co-cultured with target AML cell lines expressing the CD69 antigen, CD69 CAR-T cells showed lytic efficacy in both SHI-1 and HL60 target cell lines (Figure 31). The same results of CD69 CAR-T cells were not observed with empty CAR T cells (MOI 5, mock-transduced T cells). Consistently, significant in vitro antitumor activity against target cell lines by the four tested ScFv sequences (A1, F1, C2 and H2 in Tables 13-14) was measured, ranging between 20-90% depending on the cell line. Thus, CAR-T products A1, F1, C2 and H2 showed the desired efficacy and specificity when monitored in vitro for up to 48 hours.

[0558] CAR-T cell lytic potency is calculated as follows:

number

[0559] Each bar in Figure 31 represents the percentage of killing achieved by a different CAR-T product against an AML sample. 4.6. Example 6: In vivo efficacy of B8 and F12 CD84 ScFv(s) and H3 ScFv for CD69 CAR-T cells

[0560] Two antigens, CD84 and CD69, were investigated in vivo as targets for AML immunotherapy. CAR-T cells were produced as described in Example 3 using four different specific ScFvs identified within a human naive antigen-binding fragment (Fab) phage library, namely F12 and B8, which recognize CD84, and H3, G1, A1, F1, C2 and H2, which recognize CD69. The nucleotide sequences are provided in Figure 27. The specificity of the produced anti-CD84 B8 chain, as well as the in vivo potency of the produced CAR-T cells against CD84 (F12 and B8 chains) and CD69 (H3 chain), were examined using the SHI-1 AML cell line, which expresses high levels of both CD84 and CD69, as target cells, and against CD84-negative (CD84- The U937 (Figure 28A) and K562 (Figure 28B) cell lines, which are CD84 + CD69 + ) AML cell lines were engineered to express luciferase for non-invasive bioluminescence imaging (BLI). Manufactured CAR-T cells targeting CD84 (B8 and F12 chains) and CD69 (H3 chain) were injected into NOD / SCID gamma (NSG) mice engrafted with the targeted AML cell lines (N=7 for CD84, N=10 for CD69). Briefly, mice were injected with 0.5×10 6 AML cells / mouse were injected via the tail vein (day 1), followed by (day 3) 1.5 × 10 6 Mice were injected with CAR-T cells or mock-transduced T cells (MOI 5, empty CAR). lytic activity

[0561] The lytic activity of CD84-targeting CAR-T cells and CD69-targeting CAR-T cells is shown in Figure 28A and Figure 27B, respectively. As shown, mice injected with CAR-T cells reduced leukemic burden as measured by reduction in bioluminescence (BLI) with B8 targeting CD84 from day 24 to day 38 (*p<0.05, **p<0.005, Mann-Whitney test), F12 targeting CD84 from day 38 to day 45 (*p<0.05, Mann-Whitney test), and SHI-1 (CD84 + CD69 + ) showed a significant increase in lytic activity against AML cell lines. The increased lytic potency of mice injected with CAR-T cells is indicated by a reduction in luciferase bioluminescence signal, shown as total flux, as shown in Figures 27A-27B. In contrast, there was an increase in luciferase signal from day 24 to day 45 with empty CAR-T cells.

[0562] As shown in Figures 27C-27D, survival of mice reached 90 days after injection of CAR-T cells containing scFvs of F12, B8 to target CD84 and H3 to target CD69. This indicates superimposable in vivo efficacy of CAR T cell constructs containing scFv(s) F12, B8 or H3 to treat AML (Figures 27C-27D, *p<0.05, Mantel-Cox test). In contrast, survival of mice injected with empty CAR-T cells was approximately 49 days. 4.7. Example 7: In vivo specificity of B8 CD84 scFv CAR-T cells

[0563] To test the specificity of the new anti-CD84 B8 chain, anti-CD84 B8 CAR-T cells were cultured in U937 (CD84 neg ) and K562(CD84 neg ) into NSG mice engrafted with AML cell lines. (CD84 neg ) and K562(CD84 neg ) AML cell lines were engineered to express luciferase for non-invasive bioluminescence imaging (BLI). The manufactured CAR-T cells targeting CD84 were then transfected with two U937 (CD84 neg ) and K562(CD84 neg ) into NSG mice engrafted with an AML non-target cell line. Briefly, mice were injected with 0.5×10 6 AML cells / mouse were injected via tail vein (day 1), followed by (day 3) 1.5 × 10 6Mice were injected with 10 CAR-T cells or mock-transduced T cells (MOI 5, mCherry:mouse) (N=6-10 animals / group). As shown in Figures 28A-28B, mice injected with anti-CD84 CAR-T cells showed similar increased AML engraftment as empty CAR-T cells without reduced leukocyte load as monitored by BLI up to days 30 and 22 after challenge with K562 and U937 cells, respectively. At the time of mouse death or sacrifice, the presence of T cells was monitored by CD3 along with expression of CD84 in both bone marrow and AML-infiltrated spleen. No expression of CD84 was detected and the percentage of T cells ranged between 2-86%.

[0564] Three novel ScFv(s), one against CD69 (H3) and two against C84 (B8 and F12) in Table 12, were found to be highly specific for the target AML cell lines and showed high efficacy in vivo in NGS mice. In addition, engineered CAR-T cells containing ScFv(s) against CD69 (A1, F1, C2 or H2 in Table 12) were found to exert lytic efficacy in vitro against the target cell lines.

[0565] Five AML patient-derived xenograft (PDX) models were prepared as shown in FIG. 29 and for their high expression of three TSAs, CD84, CD69 and CD72, which are targets of the ScFv(s) of the present disclosure when expressed by CAR T cells. PDXs closely recapitulating the patient's disease fulfilled the specific demands of late-stage in vivo studies. PDX models have emerged as a fundamental preclinical tool to efficiently bridge bench and clinical data in translational research. PDX generation consisted of modeling tumors in vivo by directly transferring primary samples into immunodeficient mice, avoiding intermediate in vitro culture passaging that may induce genetic transformation or clonal selection. As a result, the findings in AML cell lines were validated and supported by the latest findings of TSA expression in AML-PDXs. In vitro cytotoxicity assay

[0566] Cytotoxicity assays were performed at the end of T cell expansion (day 17) by co-culturing AML target cell lines (positive for antigen) or ex vivo AML with CAR-T cells or non-transduced T cells for 48 hours at an E:T ratio of 1:1. Co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry with a FACSCelesta™ Cell Analyzer and FlowJo software. The percentage of cell lysis was calculated using the following formula:

number

[0567] Procedures involving animals and their care were carried out in accordance with national and international laws and policies (EEC Council Directive 86 / 609, OJ L 358, 12 December 1987) as well as institutional guidelines adhering to the "ARRIVE" guidelines (Animals in Research Reporting In Vivo Experiments). Ministerial Empowerment Approval: 131 / 2022-PR. NSG mice (4-5 week old female, 20-25 g / mouse, maximum N=5 animals / cage) were injected with 0.5 × 10 6 Two days after AML injection, mice were injected with 1.5 × 10 SHI-1-LUC (transduced with luciferase gene) cells at an E:T ratio of 3:1. 6Tumors were treated by intravenous injection of CAR-T or non-transduced / mock-transduced T cells as a control. Bioluminescence was monitored by intraperitoneal injection with XenoLight D-luciferin firefly (15 mg / ml in PBS; Perkin Elmer, Waltham, Mass.) 10 min prior to measurement (Xenogen IVIS Spectrum Bioluminescence / Optical Imaging System, Xenogen Corporation, Alameda, Calif.) to verify tumor growth. statistical analysis

[0568] Significant differences in means were tested by either the Mann-Whitney test or the Mantel-Cox test. Graphs and associated statistical analyses were generated using GraphPad Prism 8. All data are presented as mean ± standard error of the mean (SEM). Values ​​of *P<0.05, **P<0.005, ***P<0.0005, and ****P<0.0001 are statistically significant. 4.8. Example 8: In vitro cytokine production capacity of novel B8 and F12 CD84 ScFv(s) CAR-T cells

[0569] Anti-CD84 CAR-T cells were generated using two different ScFvs, namely F12 and B8, and were expressed in vitro against SHI-1 and HL60 (CD84 + and CD69 + The present inventors tested their functional ability to produce T cell-associated cytokines in response to binding of target antigens to AML cell lines (which express both T cell-associated and T cell-associated cytokines, respectively).

[0570] The HL-60 and SHI-1 AML cell lines used as target cells were grown in RPMI and DMEM (Life Technologies; 41965039; 11875093), respectively, supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106), 1% penicillin-streptomycin (P / S, 10000U / mL, Life Technologies; 15140148) and 1% L-glutamine (200mM, ThermoFisher; 25030024). ko An AML cell line was used as a control with the CD84 gene knocked out (Synthego Corporation, Redwood City, CA). All cell lines were cultured at 4 °C for 24 h at 37 °C in a 5% CO 2 Primary AML ex vivo cells were cultured at 37°C in RPMI Medium 1640 containing 10% FBS, 2 mM glutamine (Gibco, Life Technologies), 100 U / mL streptomycin / penicillin (Gibco, Life Technologies) and supplemented with 50 ng / mL thrombopoietin (TPO), 50 ng / mL stem cell factor (SCF), 50 ng / mL FMS-like tyrosine kinase 3 ligand (Flt3L), 20 ng / mL interleukin-3 (IL 3) and 20 ng / mL interleukin-6 (IL 6). All cytokines were purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, DE).

[0571] B8 or F12 CAR T cells, or CAR T cells carrying an empty vector, were cultured with target AML cell lines SHI-1 or HL60 for 48 hours at an effector to target (E:T) ratio of 1:1. Cells were then labeled for cell surface antigen expression, fixed, permeabilized, stained with anti-IFNγ and anti-TNFα antibodies, and analyzed by flow cytometry to determine the percentage of cells expressing IFNγ or anti-TNFα. Cytokine intracellular staining

[0572] After 6, 24 and 48 h of co-culture of B8 CD84 or F12 CD84 CAR-T cells with AML cells SHI-1 or HL-60 (1:1 E:T ratio), and A1 CD69 or C2 CD69 CAR T with AML cells SHI-1 or HL-60 (1:1 E:T ratio), the cytokine production capacity of B8 CD84, F12 CD84, A1 CD69 and C2 CD69 CAR-T cells was assessed by flow cytometry. Briefly, to distinguish effector CAR T cells from target AML cells, 0.4 × 10 6 Cells were labeled for cell surface antigen expression (i.e., CD45, CD3, CD33). Then, cells were fixed, permeabilized, and stained with anti-IFNγ and anti-TNFα antibodies by using the Inside Stain Kit (Miltenyi Biotec; 130-090-477) according to the manufacturer's instructions. Finally, the percentage of IFNγ and TNFα positive expressing cells relative to total lymphocytes was estimated, and data were presented as histograms with the mean + SEM of replicate measurements. result

[0573] The manufactured CAR-T cells targeting CD84 with B8 and F12 scFv chains (used interchangeably herein as "B8 CD84" and "F12 CD84" CAR-T cells) targeted the targets SHI-1 and HL60 (CD84 + / CD69 + ) AML cell lines, at an effector to target (E:T) ratio of 1:1, showed a higher frequency of positive cells expressing IFNγ and TNFα compared to CAR-T cells alone ("---") (Figure 32A and Figure 32B, respectively), thus demonstrating their ability to appropriately produce proinflammatory cytokines upon antigen binding (*p<0.05, **p<0.005, Mann-Whitney T test, n=1-4 independent experiments). 4.9. Example 9: Anti-CD84 CAR-T cells show no toxicity to CD34+ hematopoietic stem and progenitor cells (HSPCs)

[0574] Commercially available healthy donor CD34 + The in vitro off-activity of anti-CD84 CAR-T cells against hematopoietic stem / progenitor cells (HSCs) was investigated in a standard colony-forming unit (CFU) assay at an effector-to-target ratio (E:T) of 1:1. CFU assay

[0575] Commercially available CD34 from healthy donors + Cells (Hu BM CD34+, StemExpress; BM34001C) were co-cultured with either medium alone, empty CAR, B8 CD84 or F12 CD84 CAR T cells at an effector-to-target ratio of 1:1 for 4 h. After incubation, cell suspensions were added to semi-solid methylcellulose-based medium Methocult H4534 Classic without EPO (StemCell Technologies Inc, Vancouver, British Columbia, Canada) and plated in 3 cm tissue culture dishes. After 10–12 days, colonies derived from granulocyte-macrophage (G, M, and GM) and multipotent granulocyte, erythroid, macrophage, megakaryocyte (GEMM) precursors were scored and counted according to the manufacturer's instructions, and the total number of colonies was assessed. Data are presented as the mean ± SEM of independent replicate measurements. result

[0576] With constructs made with B8 and F12 scFv chains specific for the CD84 antigen, both B8 CD84 CAR T cells and F12 CD84 CAR T cells were able to express CD34 as shown by Figures 33A-33B. + B8 CD84 and F12 CD84 CAR T cells did not produce a significant effect on CD34 cells. + did not induce a reduction in the total number of CFUs formed by HSCs (Figure 33A), and CD34 +It also did not induce a reduction in colony subtypes arising from HSCs, including granulocyte-macrophage (G, M, and GM) and multipotent granulocyte, erythroid, macrophage, megakaryocyte (GEMM) precursors (FIG. 33B), supporting the finding that all hematopoietic precursors can be reconstituted (FIG. 33B). 4.10. Example 10: In vivo specificity of novel B8 CD84 ScFv CAR-T cells

[0577] To test the specificity of B8 CD84 and F12 CD84 CAR-T cells, we used SHI-1-CD84 ko AML cell lines were used as non-target cell lines due to their null CD84 expression. Specifically, the luciferase-transduced AML cell lines were engineered by a third party and purchased from Synthego Corporation to genetically knock out the CD84 antigen as confirmed by flow cytometry analysis. The manufactured B8 and F12 CD84 CAR-T cells targeting CD84 were transfected with luciferase-expressing SHI-1-CD84 ko The AML cell lines were injected into engrafted NOD / SCID gamma (NSG) mice.

[0578] Procedures involving animals and their care were carried out in accordance with national and international laws and policies (EEC Council Directive 86 / 609, OJ L 358, 12 December 1987) as well as institutional guidelines adhering to the "ARRIVE" guidelines (Animals in Research Reporting In Vivo Experiments). Ministerial Authorisation Approval: 131 / 2022-PR. NSG mice (NOD.Cg-PrkdcscidII2rgtm1Wjl / SzJ, 4-5 week old female, 20-25 g / mouse, max 5 animals / cage) were incubated with 0.5 × 10 6 Mice were injected intravenously (tail vein) with either SHI-1-LUC (transduced with luciferase gene) or HL60-LUC cells. Two days after AML injection (day 3), mice were transduced with 1.5 × 106 The mice were treated by intravenous injection of target T cells or mock-transduced T cells (empty CAR, multiplicity of infection (MOI) 5 mCherry / mouse) as a control. result

[0579] Mice injected with B8 and F12 CD84 CAR-T cells showed increased AML engraftment comparable to mice injected with empty CAR without a reduction in leukemia burden as monitored by bioluminescence imaging (BLI) from day 15 to day 45 (Figure 34A). Survival of mice injected with B8 CD84 CAR-T cells and mice injected with F12 CD84 CAR-T cells was also comparable to mice injected with empty CAR (Figure 34B). 4.11. Example 11: In vitro lytic potency of B8 CD84 and F12 CD84 CAR-T cells, and A1 CD69 and C2 CD69 CAR-T cells against primary pediatric AML ex vivo cells.

[0580] To test the activity of newly generated and manufactured CAR-T cells targeting CD84 (B8 or F12 CD84 CAR-T cells) and newly generated and manufactured CAR-T cells targeting CD69 (A1 or C2 C69 CAR-T cells), CD84 and CD69 CAR-T cells were maintained in culture alone or in co-culture with AML cells derived from AML patient-derived xenograft models (AML-PDX). AML-PDX generation

[0581] For PDX generation, NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice (4–8 weeks old) were conditioned by irradiation with 1.5 Gy 24 h prior to leukemic cell transplantation. Briefly, CD3+ T cells were prophylactically depleted by immunomagnetic cell separation (Miltenyi Biotec) using a CD3 MicroBead Kit (Miltenyi Biotec) to avoid graft-versus-host disease (GVHD). 6Primary AML cells were injected intravenously (iv) into first mouse recipients (P0). To monitor engraftment, tumor burden was assessed by flow cytometric measurement of hCD45 positive cells in peripheral blood (PB) every 2 weeks starting 1 month after AML cell injection. AML was considered engrafted when hCD45 in PB was >5%. Mice were sacrificed at 20%. Organs (femurs and spleens) were harvested and cells were harvested by flushing or mechanically dissociation and biobanked. Over two consecutive passages, 1 × 10 6 Human CD45 (hCD45) was injected intravenously into second and third recipient mice to generate P1 and P2-PDX models. The stability of the P2 model was tested by RNA and exome sequencing. Ex vivo cells were used for in vitro studies, and the P2 generation was expanded for in vivo studies. Cytotoxicity assay

[0582] Cytotoxicity assays were performed at the end of T cell expansion (day 17) by co-culturing ex vivo PDX-AML cells with CAR-T cells or empty CAR T cells at an E:T ratio of 1:1 for 48 hours. The co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry with a FACSCelesta™ Cell Analyzer and FlowJo software. The percentage of killing was calculated using the following formula:

[0583]

number

[0584] B8 and F12 CD84 (Figure 35A) and A1 and C2 CD69 (Figure 35B) CAR-T cells showed high lytic potency against primary target cells derived from three different pediatric AML-PDX models, as shown by the percent killing of AML-PDX cells after co-culture. Lytic potency ranged between 5-70% in three different AML cases (PDX #3, 6 and 7). Each bar indicated the percentage of killing made by a different CAR-T product against an AML sample.

[0585] The results show positive and consistent anti-tumor activity of B8 and F12 CD84 CAR-T cells and A1 and C2 CD69 CAR-T cells across all PDX-derived AML cells (PDX#3, PDX#6 and PDX#7 in Figures 35A-35B). 4.12. Example 12: In vitro cytokine production capacity of B8 and F12 CD84 CAR-T cells against AML-PDX cells

[0586] B8 and F12 CD84 CAR-T cells were tested for their functional ability to produce T cell-associated cytokines in response to binding to primary AML cells derived from AML-PDX in vitro. Briefly, B8 and F12 CD84 CAR T or empty CAR cells were cultured either alone (---) or with target cells derived from three different PDX-AML models at an E:T ratio of 1:1 for 48 hours, followed by labeling, fixing, and permeabilization of cells for cell surface antigen expression as described in Example 8, staining cells with anti-IFNγ and anti-TNFα antibodies, and analyzing by flow cytometry. result

[0587] B8 and F12 CD84 CAR T cells targeting CD84, when cultured with AML-PDX target cells (PDX#3, PDX#6 and PDX#7 in Figures 36A-36B), showed a higher percentage of cells positive for IFNγ and TNFα compared to when AML-PDX were cultured with empty CAR T cells (Figures 36A and 36B, respectively), thus demonstrating an enhanced ability to appropriately produce proinflammatory cytokines upon antigen binding (*p<0.05, **p<0.005, Mann-Whitney T-test, n=1-4 independent experiments). 4.13. Example 13: In vivo anti-tumor lytic activity of novel B8 CD84 ScFv CAR-T cells against AML-PDX

[0588] To test the in vivo potency and specificity of the newly generated B8 CD84 CAR T cells, one AML-PDX model derived from a pediatric patient diagnosed with AML that expresses high levels of CD84 was used.

[0589] Ten samples were collected from an AML-PDX model transduced with the luciferase gene in NSG mice. 6 AML ex vivo cells were injected via the tail vein. Mice were the...

Claims

1. An antigen-binding protein (ABP) that specifically binds to CD84, CD69, or CD84 and CD69.

2. The ABP is It contains an amino acid sequence that specifically binds to CD84 and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO:

90. It contains an amino acid sequence that specifically binds to human CD69 and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs. 89-96. The amino acid sequence specifically binds to human CD69 and CD84 and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

36. ABP as described in claim 1.

3. (i) The ABP that specifically binds to CD84, a. V having the sequence of sequence number 131 Ｌ V having the sequence CDR1, sequence number 132 Ｌ V having the sequence CDR2, sequence number 133 Ｌ V having the sequence CDR3, sequence number 251 Ｈ V having the sequence CDR1, sequence number 252 Ｈ V having the sequence CDR2 and sequence number 253 Ｈ CDR3; b. V having the sequence of SEQ ID NO: 134 Ｌ CDR1, V having the sequence of SEQ ID NO: 135 Ｌ CDR2, V having the sequence of SEQ ID NO: 136 Ｌ CDR3, V having the sequence of SEQ ID NO: 254 Ｈ CDR1, V having the sequence of SEQ ID NO: 255 Ｈ CDR2, and V having the sequence of SEQ ID NO: 256 Ｈ CDR3. c. V having the sequence of sequence number 161 Ｌ V having the sequence CDR1, sequence number 162 Ｌ V having the sequence CDR2, sequence number 163 Ｌ V having the sequence CDR3, sequence number 281 Ｈ V having the sequence CDR1, sequence number 282 Ｈ V having the sequence CDR2 and sequence number 283 Ｈ CDR3; d. V having the sequence of sequence number 164 Ｌ V having the sequence CDR1, sequence number 165 Ｌ V has the sequence CDR2, sequence number 166. Ｌ V having the sequence CDR3, sequence number 284 Ｈ V having the sequence CDR1, sequence number 285 Ｈ V having the sequence CDR2 and sequence number 286 Ｈ CDR3; e. V having the sequence of sequence number 191 Ｌ V having the sequence CDR1, sequence number 192 Ｌ V having the sequence CDR2, sequence number 193 Ｌ V having the sequence CDR3, sequence number 311 Ｈ V having the sequence CDR1, sequence number 312 Ｈ V having the sequence CDR2 and sequence number 313 Ｈ CDR3; f. V having the sequence of sequence number 194 Ｌ V having the sequence CDR1, sequence number 195 Ｌ V has the sequence CDR2, sequence number 196. Ｌ V having the sequence CDR3, sequence number 314 Ｈ V having the sequence CDR1, sequence number 315 Ｈ V having the sequence CDR2 and sequence number 316 Ｈ CDR3; g. V having the sequence of sequence number 221 Ｌ V having the sequence CDR1, sequence number 222 Ｌ V having the sequence CDR2, sequence number 223 Ｌ V having the sequence CDR3, sequence number 341 Ｈ V having the sequence CDR1, sequence number 342 Ｈ V having the sequence CDR2 and sequence number 343 Ｈ CDR3; or h. V having the sequence of sequence number 224 Ｌ V having the sequence CDR1, sequence number 225 Ｌ V having the sequence CDR2, sequence number 226 Ｌ V having the sequence CDR3, sequence number 344 Ｈ V having the sequence CDR1, sequence number 345 Ｈ V having the sequence CDR2 and sequence number 346 Ｈ CDR3 including; or (ii) The ABP that specifically binds to CD69, i. V L CDR1 having the sequence of sequence number 107, V L CDR2 having the sequence of sequence number 108, V L CDR3 having the sequence of sequence number 109, V H CDR1 having the sequence of sequence number 227, V H CDR2 having the sequence of sequence number 228, and V H CDR3 having the sequence of sequence number 229; j. V L CDR1 having the sequence of sequence number 113, V L CDR2 having the sequence of sequence number 114, V L CDR3 having the sequence of sequence number 115, V H CDR1 having the sequence of sequence number 233, V H CDR2 having the sequence of sequence number 234, and V H CDR3 having the sequence of sequence number 235; k. V L CDR1 having the sequence of sequence number 116, V L CDR2 having the sequence of sequence number 117, V L CDR3 having the sequence of sequence number 118, V H CDR1 having the sequence of sequence number 236, V H CDR2 having the sequence of sequence number 237, and V H CDR3 having the sequence of sequence number 238; l. V L CDR1 having the sequence of sequence number 119, V L CDR2 having the sequence of sequence number 120, V L CDR3 having the sequence of sequence number 121, V H CDR1 having the sequence of sequence number 239, V H CDR2 having the sequence of sequence number 240, and V H CDR3 having the sequence of sequence number 241; m. V L CDR1 having the sequence of sequence number 122, V L CDR2 having the sequence of sequence number 123, V L CDR3 having the sequence of sequence number 124, V H CDR1 having the sequence of sequence number 242, V H CDR2 having the sequence of sequence number 243, and V H CDR3 having the sequence of sequence number 244; n. V L CDR1 having the sequence of sequence number 125, V L CDR2 having the sequence of sequence number 126, V L CDR3 having the sequence of sequence number 127, V H CDR1 having the sequence of sequence number 245, V H CDR2 having the sequence of sequence number 246, and V H CDR3 having the sequence of sequence number 247; o. V L CDR1 having the sequence of sequence number 128, V L CDR2 having the sequence of sequence number 129, V L CDR3 having the sequence of sequence number 130, V H CDR1 having the sequence of sequence number 248, V H CDR2 having the sequence of sequence number 249, and V H CDR3 having the sequence of sequence number 250; p. V L CDR1 having the sequence of sequence number 137, V L CDR2 having the sequence of sequence number 138, V L CDR3 having the sequence of sequence number 139, V H CDR1 having the sequence of sequence number 257, V H CDR2 having the sequence of sequence number 258, and V H CDR3 having the sequence of sequence number 259; q. V L CDR1 having the sequence of sequence number 143, V L CDR2 having the sequence of sequence number 144, V L CDR3 having the sequence of sequence number 145, V H CDR1 having the sequence of sequence number 263, V H CDR2 having the sequence of sequence number 264, and V H CDR3 having the sequence of sequence number 265; r. V L CDR1 having the sequence of sequence number 146, V L CDR2 having the sequence of sequence number 147, V L CDR3 having the sequence of sequence number 148, V H CDR1 having the sequence of sequence number 266, V H CDR2 having the sequence of sequence number 267, and V H CDR3 having the sequence of sequence number 268; s. V L CDR1 having the sequence of sequence number 149, V L CDR2 having the sequence of sequence number 150, V L CDR3 having the sequence of sequence number 151, V H CDR1 having the sequence of sequence number 269, V H CDR2 having the sequence of sequence number 270, and V H CDR3 having the sequence of sequence number 271; t. V L CDR1 having the sequence of sequence number 152, V L CDR2 having the sequence of sequence number 153, V L CDR3 having the sequence of sequence number 154, V H CDR1 having the sequence of sequence number 272, V H CDR2 having the sequence of sequence number 273, and V H CDR3 having the sequence of sequence number 274; u. V L CDR1 having the sequence of sequence number 155, V L CDR2 having the sequence of sequence number 156, V L CDR3 having the sequence of sequence number 157, V H CDR1 having the sequence of sequence number 275, V H CDR2 having the sequence of sequence number 276, and V H CDR3 having the sequence of sequence number 277; v. V L CDR1 having the sequence of SEQ ID NO: 158, V L CDR2 having the sequence of SEQ ID NO: 159, V L CDR3 having the sequence of SEQ ID NO: 160, V H CDR1 having the sequence of SEQ ID NO: 278, V H CDR2 having the sequence of SEQ ID NO: 279, and V H CDR3 having the sequence of SEQ ID NO: 280; w. V L CDR1 having the sequence of sequence number 167, V L CDR2 having the sequence of sequence number 168, V L CDR3 having the sequence of sequence number 169, V H CDR1 having the sequence of sequence number 287, V H CDR2 having the sequence of sequence number 288, and V H CDR3 having the sequence of sequence number 289; x. V L CDR1 having the sequence of sequence number 173, V L CDR2 having the sequence of sequence number 174, V L CDR3 having the sequence of sequence number 175, V H CDR1 having the sequence of sequence number 293, V H CDR2 having the sequence of sequence number 294, and V H CDR3 having the sequence of sequence number 295; y. V L CDR1 having the sequence of sequence number 176, V L CDR2 having the sequence of sequence number 177, V L CDR3 having the sequence of sequence number 178, V H CDR1 having the sequence of sequence number 296, V H CDR2 having the sequence of sequence number 297, and V H CDR3 having the sequence of sequence number 298; z. V L CDR1 having the sequence of sequence number 179, V L CDR2 having the sequence of sequence number 180, V L CDR3 having the sequence of sequence number 181, V H CDR1 having the sequence of sequence number 299, V H CDR2 having the sequence of sequence number 300, and V H CDR3 having the sequence of sequence number 301; aa. V L CDR1 having the sequence of sequence number 182, V L CDR2 having the sequence of sequence number 183, V L CDR3 having the sequence of sequence number 184, V H CDR1 having the sequence of sequence number 302, V H CDR2 having the sequence of sequence number 303, and V H CDR3 having the sequence of sequence number 304; bb. V L CDR1 having the sequence of sequence number 185, V L CDR2 having the sequence of sequence number 186, V L CDR3 having the sequence of sequence number 187, V H CDR1 having the sequence of sequence number 305, V H CDR2 having the sequence of sequence number 306, and V H CDR3 having the sequence of sequence number 307; cc. V L CDR1 having the sequence of SEQ ID NO: 188, V L CDR2 having the sequence of SEQ ID NO: 189, V L CDR3 having the sequence of SEQ ID NO: 190, V H CDR1 having the sequence of SEQ ID NO: 308, V H CDR2 having the sequence of SEQ ID NO: 309, and V H CDR3 having the sequence of SEQ ID NO: 310; dd. V L CDR1 having the sequence of SEQ ID NO: 197, V L CDR2 having the sequence of SEQ ID NO: 198, V L CDR3 having the sequence of SEQ ID NO: 199, V H CDR1 having the sequence of SEQ ID NO: 317, V H CDR2 having the sequence of SEQ ID NO: 318, and V H CDR3 having the sequence of SEQ ID NO: 319; ee. V L CDR1 having the sequence of sequence number 203, V L CDR2 having the sequence of sequence number 204, V L CDR3 having the sequence of sequence number 205, V H CDR1 having the sequence of sequence number 323, V H CDR2 having the sequence of sequence number 324, and V H CDR3 having the sequence of sequence number 325; ff. V L CDR1 having the sequence of sequence number 206, V L CDR2 having the sequence of sequence number 207, V L CDR3 having the sequence of sequence number 208, V H CDR1 having the sequence of sequence number 326, V H CDR2 having the sequence of sequence number 327, and V H CDR3 having the sequence of sequence number 328; gg. V L CDR1 having the sequence of sequence number 209, V L CDR2 having the sequence of sequence number 210, V L CDR3 having the sequence of sequence number 211, V H CDR1 having the sequence of sequence number 329, V H CDR2 having the sequence of sequence number 330, and V H CDR3 having the sequence of sequence number 331; hh. V L CDR1 having the sequence of sequence number 212, V L CDR2 having the sequence of sequence number 213, V L CDR3 having the sequence of sequence number 214, V H CDR1 having the sequence of sequence number 332, V H CDR2 having the sequence of sequence number 333, and V H CDR3 having the sequence of sequence number 334; or ii. V L CDR1 having the sequence of sequence number 215, V L CDR2 having the sequence of sequence number 216, V L CDR3 having the sequence of sequence number 217, V H CDR1 having the sequence of sequence number 335, V H CDR2 having the sequence of sequence number 336, and V H CDR3 having the sequence of sequence number 337 including; or (iii) The ABP that specifically binds to CD84 and CD69, jj. V L CDR1 having the sequence of sequence number 45, V L CDR2 having the sequence of sequence number 52, V L CDR3 having the sequence of sequence number 55, V H CDR1 having the sequence of sequence number 63, V H CDR2 having the sequence of sequence number 66, and V H CDR3 having the sequence of sequence number 71. kk. V L CDR1 having the sequence of sequence number 110, V L CDR2 having the sequence of sequence number 111, V L CDR3 having the sequence of sequence number 112, V H CDR1 having the sequence of sequence number 230, V H CDR2 having the sequence of sequence number 231, and V H CDR3 having the sequence of sequence number 232; ll. V L CDR1 having the sequence of sequence number 140, V L CDR2 having the sequence of sequence number 141, V L CDR3 having the sequence of sequence number 142, V H CDR1 having the sequence of sequence number 260, V H CDR2 having the sequence of sequence number 261, and V H CDR3 having the sequence of sequence number 262; mm. V L CDR1 having the sequence of sequence number 170, V L CDR2 having the sequence of sequence number 171, V L CDR3 having the sequence of sequence number 172, V H CDR1 having the sequence of sequence number 290, V H CDR2 having the sequence of sequence number 291, and V H CDR3 having the sequence of sequence number 292; nn. V L CDR1 having the sequence of sequence number 200, V L CDR2 having the sequence of sequence number 201, V L CDR3 having the sequence of sequence number 202, V H CDR1 having the sequence of sequence number 320, V H CDR2 having the sequence of sequence number 321, and V H CDR3 having the sequence of sequence number 322 including, The ABP according to claim 2.

4. (i) The ABP that specifically binds to CD84, a. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 80, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 88; or b. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 81, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

88. including; or (ii) The ABP that specifically binds to CD69, a. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 85; b. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 87; c. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 83; d. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82; e. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 84; f. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 86; g. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 84; h. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 78, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

84. including; or (iii) The ABP that specifically binds to CD84 and CD69, j. A light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

83. including, ABP as described in claim 1.

5. The ABP according to claim 1, comprising an amino acid sequence selected from sequence numbers 89 to 98.

6. Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ The ABP according to claim 1, comprising a single-chain antibody molecule, a bivariable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody.

7. a. An extracellular antigen-binding domain which is an antigen-binding protein (ABP) according to claim 1, b. Transmembrane domain, c. Signal transduction domains, and d. At least one co-stimulatory domain as needed A chimeric antigen receptor (CAR) that includes this receptor.

8. The CAR according to claim 7, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) of an antibody that specifically binds to CD84, CD69, or both CD84 and CD69.

9. It further includes a co-stimulatory domain, The aforementioned co-stimulatory domain is the CD28 co-stimulatory domain, the 4-1BB co-stimulatory domain, the CD27 co-stimulatory domain, the OX40 co-stimulatory domain, or the ICOS co-stimulatory domain; The aforementioned transmembrane domain is a CD28 transmembrane domain; The aforementioned signal transduction domain is a CD3 zeta signal transduction domain. The CAR according to claim 7.

10. The CAR according to claim 7, comprising a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to an amino acid sequence selected from SEQ ID NOs. 35 to 44.

11. A polynucleotide encoding a CAR according to claim 7, wherein the polynucleotide comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity with respect to the nucleotide sequences of SEQ ID NOs. 24 to 34.

12. A vector comprising a polynucleotide as described in claim 11.

13. An immune-responsive cell expressing CAR according to any one of claims 7 to 10.

14. A composition for treating a subject, comprising immune-responsive cells expressing ABP according to any one of claims 1 to 6 or CAR according to any one of claims 7 to 10.

15. The composition according to claim 14, wherein the subject has myeloid dysplasia (MD) or acute leukemia (AL), and the myeloid dysplasia (MD) and acute leukemia (AL) are of the childhood or adult-onset type.

16. The composition according to claim 14, characterized in that the composition is administered in combination with an additional agent.

17. The composition according to claim 14, characterized in that the subject is treated with a chemotherapeutic agent or hematopoietic stem cells before the step of administering the composition.

18. The composition according to claim 14, characterized in that the step of administering the composition is not followed by, or performed in combination with, immunoglobulin therapy or autologous / allogeneic hematopoietic stem cell therapy (HSC) to aid hematopoiesis.

19. The composition according to claim 18, wherein the immunoglobulin therapy is intravenous immunoglobulin (IVIG) treatment.

20. The composition according to claim 14, wherein the subject has a refractory disease or relapse, and the subject is an adult AML patient or a pediatric AML patient.

21. The composition according to claim 14, wherein, prior to the step of administering the composition to the subject, the subject has previously been administered a chemotherapeutic agent or has previously undergone hematopoietic stem cell therapy.

22. The composition according to claim 14, wherein the subject is not responsive to either chemotherapy or hematopoietic stem cell therapy.

23. The aforementioned subject is, Myeloblastic (M0) type AML; Myeloblastic (M1) type AML; Myeloblastic (M2) type AML; Promyelocytic (M3) type AML; Myelomonocytic (M4) type AML; Monocytic (M5) type AML; AML of the erythroleukemia (M6) type; or Megakaryotic (M7) type AML The composition according to claim 14, having the following characteristics.

24. The composition according to claim 14, characterized in that the immune-responsive cells are administered to the subject in a dose ranging from 100,000 cells / kg to 25 million cells / kg.

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