BINDERS AND CHIMERIC ANTIGEN RECEPTORS WHICH SPECIFICALLY BIND FIBROBLAST GROWTH FACTOR RECEPTOR 4

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled #####, created #####, which is approximately ##### kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Some embodiments of the methods and compositions provided herein relate to chimeric antigen receptors (CARs), which specifically bind to fibroblast growth factor receptor 4 (FGFR4). Some embodiments concern nucleic acids encoding such CARs, and cells containing such CARs. Some embodiments include the use of such CARs in therapies for a cancer, such as an FGFR4-expressing cancer, including but not limited to rhabdomyosarcomas.

Some embodiments include methods and materials involved in binding a molecule, such as an antibody, a fragment of an antibody, an antibody domain, a chimeric antigen receptor (CAR), a cell engager, or an antibody-drug conjugate (ADC) to FGFR4. For example, Some embodiments include binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to an FGFR4 polypeptide and methods and materials for using such binders to treat cancer. Some embodiments include cells (e.g., host cells) designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to an FGFR4 polypeptide and methods and materials for using such cells to treat or inhibit a cancer, including but not limited to rhabdomyosarcomas.

BACKGROUND OF THE INVENTION

Rhabdomyosarcoma (RMS) is one of the most prevalent extracranial solid tumor of childhood, behind neuroblastoma and Wilms tumor (Shern J F, et al., 2015, Crit Rev Oncogenesis 20:227-243). Although significant advances have been made through the optimization of chemotherapy for early-stage disease, patients with high-risk or recurrent disease have a 5-year survival rate of <30% and 17%, respectively. Key genetic alterations seen in this disease include: a characteristic PAX3-FOXO1 or PAX7-FOXO1 fusion gene product; mutations in TP53, RAS, PI3K3CA, CTNNB1 (beta-catenin), FGFR4 (fibroblast growth factor receptor 4); and loss of heterozygosity at 11p15.5 (Li F P, et al., 1969, J Natl Cancer Inst 43:1365-1373; Steenman M, et al., 2000, Gene Chromosome Cancer 28:1-13; Barr F G, et al., 1993, Nat Genet 3:113-117; Taylor A C, et al., 2000, Med Pediatr Oncol 35:96-103; Stratton M R, et al., 1989, Cancer Res 49:6324-6327; Shukla M, et al., 2012, Clin Canc Res 18:748-757; Taylor J G et al., 2009, J Clin Invest 119:3395-3407). Historically RMS was defined by lesion morphology (alveolar, ARMS, versus embryonal, ERMS), but it has now become clear that the biology of the disease is closely associated with genotype. RMS is now defined by the presence or absence of PAX3/7 fusion genes (Shern J F, et al., 2014, Cancer Disc 4:216-231; Chen X, et al., 2013, Cancer Cell 24:710-724; Kohsaka S, et al., 2014, Nat Genet 46:595-600). Clinical studies have established that fusion gene status is more relevant to outcome in low and intermediate risk disease, than traditional pathological classification as ERMS or ARMS (Skapek S X, et al., 2013, Pediatr Blood Cancer 60:1411-1417). However, clinical studies have also established that in advanced metastatic disease, standard clinical risk factors, such as the degree of dissemination, better define outcome, which is uniformly poor and unimproved in decades (Rudzinski E R, et al., 2017, Pediatr Blood Cancer, 64:e26645).

The recent success of using immune checkpoint inhibitors to treat melanoma and lung cancer has had little impact in pediatric cancer because of the low mutational rate of pediatric tumors (Alexandrov B, et al., 2013, Nature 500:415-421). In fact, PAX gene translocation-expressing RMS has a remarkably low mutation rate, 0.1 protein coding mutations per Mb, with no recurring point mutations. Therefore, there is a need for additional improved therapies to treat or inhibit the progressions of cancers, such as RMS.

SUMMARY OF THE INVENTION

Some embodiments provided herein include methods and materials involved in binding a molecule (e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC) to an FGFR4 polypeptide. For example, some embodiments include binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to an FGFR4 polypeptide and methods and materials for using one or more such binders in a therapy for a mammal (e.g., a human) having cancer, including but not limited to rhabdomyosarcomas.

Some embodiments include cells (e.g., host cells) designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to an FGFR4 polypeptide and methods and materials for using such cells to treat or inhibit a cancer, including but not limited to rhabdomyosarcomas.

As described herein, binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more CARs, one or more cell engagers, and/or one or more ADCs) can be designed to have the ability to bind to an FGFR4 polypeptide. For example, a binder (e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC) provided herein can have the ability to bind to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human FGFR4.

In some cases, a single set of three complementarity-determining regions (CDRs) of an antibody domain (e.g., a VH domain) provided herein, for example, such as in TABLE 3 and TABLE 4, or two sets of three CDRs of an antibody (e.g., scFc) provided herein, for example, such as in TABLE 3 and TABLE 4, can be engineered into a CAR to create CAR⁺ cells (e.g., CAR⁺ T cells, CAR⁺ stem cells such as CAR⁺ induced pluripotent stem cells, or CAR⁺ natural killer (NK) cells) having the ability to target FGFR4⁺ cells (e.g., FGFR4⁺ tumor cells and/or FGFR4⁺ tumor vasculature), can be engineered into an antibody structure that includes an Fc region to create antibodies having the ability to target FGFR4⁺ cells (e.g., FGFR4⁺ tumor cells and/or FGFR4⁺ tumor vasculature) and induce antibody-dependent cell-mediated cytotoxicity (ADCC) against the target FGFR4 cells, and/or can be engineered into a cell engager such as a bi-specific T cell engager (e.g., a BiTE), a bi-specific killer engager (e.g., a BiKE), and/or a tri-specific killer engager (e.g., a TriKE) to create cell engagers having the ability to target FGFR4⁺ cells (e.g., FGFR4⁺ tumor cells and/or FGFR4⁺ tumor vasculature) and induce one or more immune responses (e.g., T cell immune responses and/or ADCC using a cell engager in the absence of an Fc-containing antibody) against the target FGFR4⁺ cells. It is noted that BiKE- and TriKE-mediated killing can be referred to ADCC even though it is not initiated by an Fc domain.

In addition, as described herein, binders (e.g., one or more antibodies, one or more antigen binding fragments, and/or one or more antibody domains) provided herein can be used to create conjugates that include the binder and a drug. For example, ADCs such as full antibody-drug conjugates, Fab-drug conjugates, and/or antibody domain-drug conjugates can be designed to include an appropriate binder provided herein to create the conjugate. Such conjugates can be used to deliver the drug payload to target cells such as cancer cells (e.g., FGFR4⁺ cancer cells) or cancer vasculature (e.g., FGFR4⁺ cancer vasculature).

As also described herein, binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein can be used in a therapy for a mammal (e.g., a human) having cancer, including but not limited to rhabdomyosarcomas. For example, a mammal (e.g., a human) having cancer (e.g., an FGFR4⁺ cancer, including but not limited to rhabdomyosarcomas) can be administered a composition comprising one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) described herein to reduce the number of cancer cells within the mammal, to induce ADCC against cancer cells within the mammal, and/or to increase the survival duration of the mammal from the cancer.

As also described herein, cells (e.g., host cells) can be designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to an FGFR4 polypeptide. For example, cells such as T cells (e.g., CTLs), stem cells (e.g., induced pluripotent stem cells), or NK cells can be engineered to express one or more CARs having the ability to bind to an FGFR4 polypeptide. Such cells (e.g., FGFR4-specific CAR⁺ T cells or NK cells) can be used to treat or inhibit a cancer, including but not limited to rhabdomyosarcomas.

In some embodiments, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be used to detect the presence or absence of an FGFR4 polypeptide. For example, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be used to determine whether or not a sample (e.g., a biological sample such tumor biopsy) obtained from a mammal (e.g., a human) contains FGFR4⁺ cells (e.g., FGFR4⁺ cancer cells). Having the ability to detect the presence or absence of an FGFR4 polypeptide (e.g., FGFR4⁺ cancer cells) can allow clinicians, health professionals, and patients to make better decisions about possible treatment options. For example, detection of FGFR4⁺ cancer cells within a mammal can allow clinicians, health professionals, and patients to select an appropriate anti-cancer treatment that targets the FGFR4 cancer cells. Such therapies that target the FGFR4⁺ cancer cells can include administration of an anti-FGFR4 antibody such as U3-1784 and/or one or more of the binders described herein having the ability to bind to an FGFR4 polypeptide and/or administration of one or more cells (e.g., FGFR4-specific CAR⁺ T cells or NK cells) designed to express a binder described herein.

Some embodiments of the methods and compositions provided herein include a nucleic acid comprising a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: a ligand binding domain capable of specifically binding or configured to specifically bind to an IgM membrane-proximal domain of a fibroblast growth factor receptor 4 (FGFR4) protein; a transmembrane domain; and an intracellular signalling domain.

In some embodiments, the ligand binding domain comprises a complementarity-determining region (CDR). In some embodiments, the ligand binding domain comprises a heavy chain complementarity-determining region (CDR). In some embodiments, the ligand binding domain comprises a first additional heavy chain CDR. In some embodiments, the ligand binding domain comprises a second additional heavy chain CDR. In some embodiments, the ligand binding domain comprises a light chain CDR.

In some embodiments, the CDR comprises an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:42-149. In some embodiments, the CDR comprises an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:41-53, 57-62, 78-101, 114-125 and 132-137. In some embodiments, the CDR comprises an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:57-59.

In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:01-22. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:01-04, 06, 07, 11-14, 17, 18 and 20. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:06. In some embodiments, the ligand binding domain comprises the amino acid sequence of SEQ ID NO:06.

In some embodiments, the ligand binding domain comprises, consists essentially of, or consists of a variable heavy chain (VH) domain. In some embodiments, the ligand binding domain lacks a variable light chain (VL) domain.

In some embodiments, the transmembrane domain comprises a domain selected from a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:24.

In some embodiments, the CAR comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:41.

In some embodiments, the CAR further comprises a spacer located between the ligand binding domain and the transmembrane domain.

Some embodiments also include a constitutive promoter operably linked to the polynucleotide encoding the CAR. In some embodiments, the constitutive promoter comprises an EF1α promoter.

Some embodiments also include an inducible promoter operably linked to the polynucleotide encoding the CAR

Some embodiments also include a polynucleotide encoding a cell-surface selectable marker. In some embodiments, the cell-surface selectable marker is selected from a truncated EGFR polypeptide (EGFRt) or a truncated Her2 polypeptide (Her2t).

Some embodiments also include a ribosome skip sequence located between a polynucleotide encoding the intracellular signalling domain and the polynucleotide encoding a cell-surface selectable marker. In some embodiments, the ribosome skip sequence is selected from the group consisting of a P2A sequence, a T2A sequence, an E2A sequence, and an F2A sequence.

Some embodiments also include a polynucleotide encoding a suicide gene system. In some embodiments, the suicide gene system is selected from a herpes simplex virus thymidine kinase/ganciclovir (HSVTK/GCV) suicide gene system, or an inducible caspase suicide gene system.

Some embodiments include a polypeptide encoded by any one of the foregoing nucleic acids.

Some embodiments include a vector comprising any one of the foregoing nucleic acids. In some embodiments, the vector is selected from the group consisting of a viral vector, a transposon vector, an integrase vector, and an mRNA vector. In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, an adeno-associated virus, a foamy viral vector, a retroviral vector, and a gamma retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

Some embodiments include a host cell comprising any one of the foregoing nucleic acids. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a CD4+ T-cell or a CD8+ T-cell. In some embodiments, the host cell is a CD8+ cytotoxic T-cell selected from the group consisting of a naïve CD8+ T-cell, a CD8+ memory T-cell, a central memory CD8+ T-cell, a regulatory CD8+ T-cell, an IPS derived CD8+ T-cell, an effector memory CD8+ T-cell, and a bulk CD8+ T-cell.

In some embodiments, the host cell is a CD4+ T helper cell selected from the group consisting of a naïve CD4+ T-cell, a CD4+ memory T-cell, a central memory CD4+ T-cell, a regulatory CD4+ T-cell, an IPS derived CD4+ T-cell, an effector memory CD4+ T-cell, and a bulk CD4+ T-cell. In some embodiments, the host cell is a precursor T-cell, or a hematopoietic stem cell. In some embodiments, the cell is allogenic to a subject. In some embodiments, the cell is human.

Some embodiments include a pharmaceutical composition comprising any one of the foregoing host cells and a pharmaceutically acceptable excipient.

Some embodiments include a method for preparing a population of cells, comprising: (a) introducing any one of the foregoing nucleic acids into an isolated population of cells; and (b) culturing the population of cells in the presence of an agent selected from an anti-CD3, an anti-CD28, and a cytokine such as IL-2. In some embodiments, the population of cells comprises a CD4+ T-cell or a CD8+ T-cell. In some embodiments, the population of cells comprises a CD8+ cytotoxic T-cell selected from the group consisting of a naïve CD8+ T-cell, a CD8+ memory T-cell, a central memory CD8+ T-cell, a regulatory CD8+ T-cell, an IPS derived CD8+ T-cell, an effector memory CD8+ T-cell, and a bulk CD8+ T-cell. In some embodiments, the population of cells comprises a CD4+ T helper cell selected from the group consisting of a naïve CD4+ T-cell, a CD4+ memory T-cell, a central memory CD4+ T-cell, a regulatory CD4+ T-cell, an IPS derived CD4+ T-cell, an effector memory CD4+ T-cell, and a bulk CD4+ T-cell. In some embodiments, the population of cells comprises a precursor T-cell, or a hematopoietic stem cell.

Some embodiments include a method of treating, inhibiting or ameliorating a cancer, including but not limited to rhabdomyosarcomas, in a subject, comprising: administering any one of the foregoing host cells to the subject.

In some embodiments, the cancer comprises an FGFR4-expressing cell. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a cell expressing a protein selected from a PAX3-FOXO1 fusion gene product, and a PAX7-FOXO1 fusion gene product. In some embodiments, the cancer comprises a cell comprising a mutation in a gene selected from TP53, RAS, PI3K3CA, CTNNB1, and FGFR4. In some embodiments, the cancer comprises a rhabdomyosarcoma. In some embodiments, the subject is human.

Some embodiments include any one of the foregoing host cells for use in treating, inhibiting or ameliorating a cancer in a subject.

Some embodiments include use of any one of the host cells in the manufacture of a medicament for treating, inhibiting or ameliorating a cancer in a subject.

Some embodiments include any one of the foregoing host cells for use as a medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts graphs for cytolytic activity in T cells expressing anti-FGFR4 CARs. T cells containing anti-FGFR4 CARs were tested against a CD19+/FGFR4⁺ cell line, RH30_19 (left panel), or a CD19(−)/FGFR4⁺ cell line, RMS559 (right panel). Specific lysis and E:T ratios are listed on the axes. Test anti-FGFR4 CARs and a control murine MAb-derived binder BT53-CAR are listed in the legend.

FIG. 2 depicts an analysis for in vivo activity of anti-FGFR4 CAR T cells in a xenograft model. NSG mice were inoculated i.v. with Rh30_19 (expressing FFLuc) and 3 days later (following imaging, post i.p injection of luciferin, on an I^VIS system) inoculated i.v. with CAR T-cells (Tumor+CAR), untransduced T cells (Tumor+UTD), or no T cells (Tumor Only). Weekly images were captured, with scaling changed on day 32. By day 59 all mice that did not receive FGFR4-specific CARs were sacrificed per protocol, while mice that did were alive on day 66.

FIG. 3 depicts an analysis of Rh30_19-bearing mice treated with M410-CAR-T. NSG mice were injected i.m. with tumor in the hind limb and subsequently untreated (left column, Tumor only), treated with M410-CAR-T (center column, CAR T cells), or infused with untransduced T cells (right column, UTD). Tumors were excised, fixed, sectioned and stained for human CD8a, CD4, or with H&E, rows 2, 3, and 1, respectively.

FIG. 4 is a schematic of a FGFR4 protein with extracellular domains including: an IgI domain, an acidic box, an IgII domain, and a membrane-proximal IgIII domain; a transmembrane domain; and intracellular domains including: a TK1 domain, and a TK2 domain.

FIG. 5 is a schematic of polynucleotides encoding anti-FGFR4 CARs and depicts certain regions encoding: a ligand binding domain (binder), such as an FGFR4-binding polypeptide; a CD8 transmembrane domain and linker domain; a 4-1BB domain; and a CD3 zeta domain. Some polynucleotides also included a promoter, and lentiviral vector sequences such as 5′ LTR, and 3′ SIN-LTR A ligand binding domain (binder) can be derived from a VH domain polypeptide, a polypeptide containing a VH domain linked to a VL domain via a linker, or a polypeptide containing a VL domain linked to a VH domain via a linker.

FIG. 6 depicts graphs of relative levels of expression of CARs in transduced T-cells. Upper panel depicts relative levels of expression of CARs containing various VH domain only FGFR4-binding polypeptides; middle panel depicts relative levels of expression of CARs containing various VL-VH domain FGFR4-binding polypeptides; and lower panel depicts relative levels of expression of CARs containing various VH-VL domain FGFR4-binding polypeptides.

FIG. 7 depicts graphs of percentage specific lysis of target cells for various ratios of effector:target cells. Effector cells included anti-FGFR4 CAR T-cells containing different CARs. Target cells included RH30 cells, and Raji cells.

FIG. 8 depicts graphs of interferon gamma production from anti-FGFR4 CAR T-cells containing different CARs incubated with Rh30 cells, which is a rhabdomyosarcoma cell line.

FIG. 9 depicts exemplary linker amino acid sequences for scFv's, CARs, and/or cell engagers, including SEQ ID NOS:244-266.

FIG. 10 depicts the amino acid sequences of exemplary hinges that can be used to design a CAR, including SEQ ID NOS:267-275.

FIG. 11 depicts the amino acid sequences of exemplary transmembrane domains that can be used to design a CAR, including SEQ ID NOS:276-283.

FIG. 12 depicts the amino acid sequences of exemplary intracellular signaling domains that can be used to design a CAR, including SEQ ID NOS:284-291.

FIG. 13 depicts the amino acid sequences of exemplary antigen binding domains that can be used to design cell engagers that bind to T cells, including SEQ ID NOS:292-295.

FIG. 14 depicts the amino acid sequences of exemplary antigen binding domains that can be used to design cell engagers that bind to NK cells, including SEQ ID NOS:296-309.

FIG. 15A is a schematic of an exemplary BiTE designed using CDR1, CDR2, and CDR3 of a VH domain provided herein. A humanized anti-CD3 scFv (e.g., an gOKT3-7 scFv set forth in U.S. Pat. No. 6,750,325, herein expressly incorporated by reference in its entirety) can be linked to the C-terminus of the VH domain via a linker (e.g., a (G4S)3 linker). FIG. 15B depicts an amino acid of a linker sequence (SEQ ID NO:310) followed by an gOKT3-7 scFv sequence (SEQ ID NO:311), which can be attached to a VH domain as shown in FIG. 15A. FIG. 15B also depicts a nucleic acid sequence encoding that linker and gOKT3-7 scFv.

DETAILED DESCRIPTION

Some embodiments provided herein include binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). For example, some embodiments include binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a polypeptide comprising, consisting essentially of, or consisting of the amino acid of human FGFR4. In some cases, a binder (e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC) provided herein can have the ability to bind to an FGFR4 polypeptide. For example, a binder (e.g., an antibody, an antigen binding fragment, an antibody domain, a CAR, a cell engager, or an ADC) provided herein can have the ability to bind to a human FGFR4 polypeptide.

The term “antibody” as used herein includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multi-specific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, single-chain variable fragment antibodies (e.g., scFv antibodies), and tandem single-chain variable fragments antibody (e.g., taFv). A diabody can include two chains, each having a heavy chain variable domain and a light chain variable domain, either from the same or from different antibodies (see, e.g., Hornig and Farber-Schwarz, Methods Mol. Biol., 907:713-27 (2012); and Brinkmann and Kontermann, MAbs., 9(2):182-212(2017)). The two variable regions can be connected by a polypeptide linker (e.g., a polypeptide linker having five to ten residues in length or a polypeptide linker as set forth in FIG. 9). In some cases, an interdomain disulfide bond can be present in one or both of the heavy chain variable domain and light chain variable domain pairs of the diabody. A scFv is a single-chain polypeptide antibody in which the heavy chain variable domain and the light chain variable domain are directly connected or connected via a polypeptide linker (e.g., a polypeptide linker having eight to 18 residues in length or a polypeptide linker as set forth in FIG. 9). See, also, Chen et al., Adv. Drug Deliv. Rev., 65(10):1357-1369 (2013). A scFv can be designed to have an orientation with the heavy chain variable domain being followed by the light chain variable domain or can be designed to have an orientation with the light chain variable domain being followed by the heavy chain variable domain. In both cases, the optional linker can be located between the two domains.

An antibody provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be configured to be a human antibody, a humanized antibody, or a chimeric antibody. In some cases, an antibody provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be a monoclonal antibody. In some cases, an antibody provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be configured as a scFv antibody.

The term “antigen binding fragment” as used herein refers to a fragment of an antibody (e.g., a fragment of a humanized antibody, a fragment of a human antibody, or a fragment of a chimeric antibody) having the ability to bind to an antigen. Examples of antigen binding fragments include, without limitation, Fab, Fab′, or F(ab′)₂antigen binding fragments. An antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be configured to be a human antigen binding fragment, a humanized antigen binding fragment, or a chimeric antigen binding fragment. In some cases, an antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be a monoclonal antigen binding fragment. In some cases, an antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be configured as a Fab antibody.

The term “antibody domain” as used herein refers to a domain of an antibody such as a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain) in the absence of one or more other domains of an antibody. In some cases, an antibody domain can be a single antibody domain (e.g., a VH domain or a VL domain) having the ability to bind to an antigen. An antibody domain provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be a human antibody domain (e.g., a human VH domain), a humanized antibody domain (e.g., a humanized VH domain), or a chimeric antibody domain (e.g., a chimeric VH domain). In some cases, an antibody domain provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be a monoclonal antibody domain. In some cases, an antibody domain provided herein can include the CDRs as described herein (e.g., as described in TABLE 3 or 4) and can be engineered as a single VH domain or a single VL domain.

An anti-FGFR4 antibody, anti-FGFR4 antigen binding fragment, or anti-FGFR4 antibody domain provided herein can be of the IgA-, IgD-, IgE-, IgG-, or IgM-type, including IgG- or IgM-types such as, without limitation, IgG₁-, IgG₂-, IgG₃-, IgG₄-, IgM₁-, and IgM₂-types. In some cases, an antibody provided herein (e.g., an anti-FGFR4 antibody) can be a scFv antibody. In some cases, an antigen binding fragment provided herein (e.g., an anti-FGFR4 antibody fragment) can be a Fab. In some cases, an antibody provided herein (e.g., an anti-FGFR4 antibody) can be a fully intact antibody consisting of both VH and VL. In some cases, an antibody domain provided herein (e.g., an anti-FGFR4 antibody domain) can be a VH domain.

As described herein, a chimeric antigen receptor can refer to a chimeric polypeptide that is designed to include an antigen binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains. As described herein, the antigen binding domain of a CAR provided herein can be designed to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). For example, a CAR provided herein can be designed to include the components of an antibody, antigen binding fragment, and/or antibody domain described herein (e.g., a combination of CDRs) as an antigen binding domain provided that that antigen binding domain has the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). In some examples, a CAR provided herein can be designed to include an antigen binding domain that includes a single set of three CDRs (e.g., a CDR1, CDR2, and CDR3) of an antibody domain (e.g., a VH domain) provided herein (see, e.g., TABLE 3 and TABLE 4). In some examples, a CAR provided herein can be designed to include an antigen binding domain that includes a first set of three CDRs (e.g., a CDR1, CDR2, and CDR3 of a heavy chain) and a second set of CDRs (e.g., a CDR1, CDR2, and CDR3 of a light chain) of an antibody (e.g., a Fab) provided herein (see, e.g., TABLE 3 and TABLE 4). In some cases, an antigen binding domain of a CAR targeting an FGFR4 polypeptide can be designed to include a VH domain described herein or a scFv antibody described herein.

In some cases, a CAR provided herein can be designed to include a hinge. Any appropriate hinge can be used to design a CAR described herein. Examples of hinges that can be used to make a CAR described herein include, without limitation, Ig-derived hinges (e.g., an IgG1-derived hinge, an IgG2-derived hinge, or an IgG4-derived hinge), Ig-derived hinges containing a CD2 domain and a CD3 domain, Ig-derived hinges containing a CD2 domain and lacking a CD3 domain, Ig-derived hinges containing a CD3 domain and lacking a CD2 domain, Ig-derived hinges lacking a CD2 domain and lacking a CD3 domain, CD8α-derived hinges, CD28-derived hinges, and CD3ζ-derived hinges. A CAR provided herein can be designed to include a hinge of any appropriate length. For example, a CAR provided herein can be designed to include a hinge that is from about 3 to about 75 (e.g., from about 3 to about 65, from about 3 to about 50, from about 5 to about 75, from about 10 to about 75, from about 5 to about 50, from about 10 to about 50, from about 10 to about 40, or from about 10 to about 30) amino acid residues in length. In some cases, a linker sequence can be used as hinge to make a CAR described herein. For example, any one of the linker sequences set forth in FIG. 9 can be used as a hinge of a CAR described herein.

In some cases, a CAR provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10. In some cases, a CAR provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a CAR provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.

A CAR provided herein can be designed to include any appropriate transmembrane domain. For example, the transmembrane domain of a CAR provided herein can be, without limitation, a CD3ζ transmembrane domain, a CD4 transmembrane domain, a CD8a transmembrane domain, a CD28 transmembrane domain, and a 4-1BB transmembrane domain. In some cases, a CAR provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 11. In some cases, a CAR provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 11 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a CAR provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 11 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.

A CAR provided herein can be designed to include one or more intracellular signaling domains. For example, a CAR provided herein can be designed to include one, two, three, or four intracellular signaling domains. Any appropriate intracellular signaling domain or combination of intracellular signaling domains can be used to make a CAR described herein. Examples of intracellular signaling domains that can be used to make a CAR described herein include, without limitation, CD3ζ intracellular signaling domains, CD27 intracellular signaling domains, CD28 intracellular signaling domains, OX40 (CD134) intracellular signaling domains, 4-1BB (CD137) intracellular signaling domains, CD278 intracellular signaling domains, DAP10 intracellular signaling domains, and DAP12 intracellular signaling domains. In some cases, a CAR described herein can be designed to be a first-generation CAR having a CD3ζ intracellular signaling domain. In some cases, a CAR described herein can be designed to be a second-generation CAR having a CD28 intracellular signaling domain followed by a CD3ζ intracellular signaling domain. In some cases, a CAR described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an OX40 intracellular signaling domains, or a 4-1BB intracellular signaling domain followed by (c) a CD3ζ intracellular signaling domain. In some cases, a CAR provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12. In some cases, a CAR provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, a CAR provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling.

The term “cell engager” as used herein refers to a polypeptide that includes two or more antigen binding domains (e.g., two, three, or four antigen binding domains) and has the ability to link two cells together. Examples of cell engagers include, without limitation, BiTEs, BiKEs, and TriKEs. In general, a cell engager provided herein can be designed to include at least one antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and at least one antigen binding domain having the ability to bind to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell). In some cases, a cell engager described herein can link an FGFR4⁺ cell (e.g., an FGFR4⁺ cancer cell) to another cell (e.g., a T cell or an NK cell) via the two or more antigen binding domains of the cell engager.

When a cell engager includes an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and two or more other antigen binding domains (e.g., two, three, or four other antigen binding domains), each of those other antigen binding domains can bind to different antigens expressed on the surface of different cell types or can bind to different antigens expressed on the surface of the same cell type. For example, a TriKE can be designed to have a first antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide), a second antigen binding domain having the ability to bind to a first antigen expressed on the surface of an NK cell (e.g., a CD16 polypeptide such as a CD16a polypeptide), and a third antigen binding domain having the ability to bind to a second antigen expressed on the surface of an NK cell (e.g., an NKG2A polypeptide).

As described herein, at least one antigen binding domain of a cell engager provided herein can be designed to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). For example, a cell engager provided herein can be designed to include the components of an antibody, antigen binding fragment, and/or antibody domain described herein (e.g., a combination of CDRs) as an antigen binding domain provided that that antigen binding domain has the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). In some examples, a cell engager provided herein can be designed to include an antigen binding domain that includes a single set of three CDRs (e.g., a CDR1, CDR2, and CDR3) of an antibody domain (e.g., a VH domain) provided herein (see, e.g., TABLE 3 or 4). In some examples, a cell engager provided herein can be designed to include an antigen binding domain that includes a first set of three CDRs (e.g., a CDR1, CDR2, and CDR3 of a heavy chain) of an antibody domain (e.g., a Fab) provided herein and a second set of three CDRs (e.g., a CDR1, CDR2, and CDR3 of a light chain) of an antibody domain (e.g., a Fab) provided herein (see, e.g., TABLE 3 or 4). In some cases, an antigen binding domain of a cell engager targeting an FGFR4 polypeptide can be designed to include a VH domain described herein. In some cases, an antigen binding domain of a CAR described herein that has the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can be used as an antigen binding domain of a cell engager that targets FGFR4⁺ cells.

As described herein, a cell engager can be designed to include at least one antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and at least one other antigen binding domain. That at least one other antigen binding domain can have the ability to bind to any appropriate antigen expressed on the surface of a cell. For example, when designing a cell engager such as a BiTE to link an FGFR4⁺ cell and a T cell, the cell engager can include an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell. Examples of polypeptides expressed on the surface of a T cell that can be targeted by an antigen binding domain of a cell engager provided herein include, without limitation, CD3 polypeptides. Examples of antigen binding domains having the ability to bind to a polypeptide expressed on the surface of a T cell that can be used to make a cell engager provided herein (e.g., a BiTE) include, without limitation, anti-CD3 scFvs and anti-CD3 VH domains. Additional examples of amino acid sequences that can be used as antigen binding domains having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., CD3) are described in U.S. Pat. No. 6,750,325, herein expressly incorporated by reference (see, e.g., the sequence listing of U.S. Pat. No. 6,750,325). In some cases, a cell engagers designed to have the ability to bind to an FGFR4 polypeptide and a T cell can have the structure as shown in FIGS. 15A and 15B.

In some cases, a cell engager provided herein can be designed to include an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 13. In some cases, a cell engager provided herein can be designed to include an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 13 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of a T cell. In some cases, a cell engager provided herein can be designed to include an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 13 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of a T cell.

When designing a cell engager such as a BiKE or a TriKE to link an FGFR4⁺ cell and an NK cell, the cell engager can include an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and one or more (e.g., one, two, or three) antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell. Examples of polypeptides expressed on the surface of an NK cell that can be targeted by an antigen binding domain of a cell engager provided herein include, without limitation, CD16 polypeptides (e.g., CD16a polypeptides), NKG2A polypeptides, NKG2D polypeptides, NKp30 polypeptides, NKp44 polypeptides, and NKp46 polypeptides. Examples of antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell that can be used to make a cell engager provided herein (e.g., a BiKE or TriKE) include, without limitation, anti-CD16a scFvs, anti-NKG2A scFvs, anti-NKG2D scFvs, anti-NKp30 scFvs (see, e.g., BioLegend Catalog #325207), anti-NKp44 scFvs, anti-NKp46 scFvs, anti-CD16a VH domains, anti-NKG2A VH domains, anti-NKG2D VH domains, anti-NKp30 VH domains, anti-NKp44 VH domains, or anti-NKp46 VH domains. Additional examples of amino acid sequences that can be used as antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., CD16, NKG2A, NKG2D, or NKp46) are described in McCall et al. (Mol. Immunol., 36(7):433-445 (1999); see, e.g., anti-CD16 scFv sequences); International Patent Application Publication No. PCT/US2017/048721 (see, e.g., the CDRs and sequence listing for anti-CD16a binding domains); U.S. Patent Application Publication No. 2011/0052606 (see, e.g., the CDRs and the sequence listing for anti-NKG2A antibodies such as Z199); U.S. Patent Application Publication No. 2011/0150870 (see, e.g., the CDRs and sequence listing for anti-NKG2D antibodies); U.S. Patent Application Publication No. 2018/0369373 (see, e.g., the CDRs and sequence listing for anti-NKp46 antibodies); and U.S. Patent Application Publication No. 2017/0368169 (see, e.g., the CDRs and sequence listing for anti-NKp46 antibodies), all of which are expressly incorporated by reference in their entireties.

In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a scFv or VH) that comprises, consists essentially of, or consists of one or more of the amino acid sequences set forth in FIG. 14. In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 14 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of an NK cell. In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 14 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of an NK cell.

In some cases, a cell engager provided herein can be designed to include a linker located between each antigen binding domain. Any appropriate linker can be used to design a cell engager provided herein. Examples of linkers that can be used to make a cell engager described herein include, without limitation, the linker sequences set forth in FIG. 9. A cell engager provided herein can be designed to include a linker of any appropriate length. For example, a cell engager provided herein can be designed to include a linker that is from about 3 to about 100 (e.g., from about 3 to about 90, from about 3 to about 80, from about 3 to about 70, from about 3 to about 60, from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 3 to about 20, from about 3 to about 15, from about 5 to about 100, from about 10 to about 100, from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 50 to about 100, from about 60 to about 100, from about 70 to about 100, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, or from about 12 to about 17) amino acid residues in length. In some cases, a cell engager provided herein (e.g., a BiTE) can be designed to include a GGGGSGGGGSGGGGS linker. In some cases, a hinge of a CAR described herein can be used as a linker to make a cell engager described herein. For example, any one of the sequences set forth in FIG. 10 can be used as a linker of a cell engager described herein.

In some cases, a cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10. In some cases, a cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 9 or FIG. 10 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.

In one embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:42 (or a variant of SEQ ID NO:42 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:43 (or a variant of SEQ ID NO:43 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:44 (or a variant of SEQ ID NO:44 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:01. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:01. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:01.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:01, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:42, 43, and 44. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:01, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:42, 43, and 44.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:01 or the amino acid set forth in SEQ ID NO:01 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:01 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:42, 43, and 44.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:42” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:42, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:42, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:42, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:43” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:43, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:43, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:43, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:44” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO. 44, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:44, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:44, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:45 (or a variant of SEQ ID NO:45 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:46 (or a variant of SEQ ID NO:46 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:47 (or a variant of SEQ ID NO:47 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:02. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:02. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:02.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:02, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:45, 46, and 47. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:02, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:45, 46, and 47.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:02 or the amino acid set forth in SEQ ID NO:02 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:02 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:45, 46, and 47.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:45, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:46, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:47. As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:45” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:45, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO. 45, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:45, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:46” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:46, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:46, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:46, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:47” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO. 47, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:47, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:47, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:48 (or a variant of SEQ ID NO:48 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:49 (or a variant of SEQ ID NO:49 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:50 (or a variant of SEQ ID NO:50 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:03. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:03. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:03.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:03, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:48, 49, and 50. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:03, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:48, 49, and 50.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:03 or the amino acid set forth in SEQ ID NO:03 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:03 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:48, 49, and 50.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:48” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:48, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:48, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:48, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:49” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:49, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:49, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:49, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:50” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:50, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:50, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:50, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:51 (or a variant of SEQ ID NO:51 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:52 (or a variant of SEQ ID NO:52 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:53 (or a variant of SEQ ID NO:53 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:04. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:04. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:04.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:04, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:51, 52, and 53. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:04, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:51, 52, and 53.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:04 or the amino acid set forth in SEQ ID NO:04 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:04 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:51, 52, and 53.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:51” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO. 51, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:51, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:51, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:52” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:52, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:52, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:52, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:53” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:53, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:53, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:53, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:54 (or a variant of SEQ ID NO:54 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:55 (or a variant of SEQ ID NO. 55 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:56 (or a variant of SEQ ID NO:56 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:05. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:05. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:05.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:05, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:54, 55, and 56. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:05, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:54, 55, and 56.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:05 or the amino acid set forth in SEQ ID NO:05 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:05 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:54, 55, and 56.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:54” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:54, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:54, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:54, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:55” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:55, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:55, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:55, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:56” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:56, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:56, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:56, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:57 (or a variant of SEQ ID NO:57 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:58 (or a variant of SEQ ID NO:58 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:59 (or a variant of SEQ ID NO:59 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:06. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:06. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:06.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:06, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:57, 58, and 59. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:06, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:57, 58, and 59.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:06 or the amino acid set forth in SEQ ID NO:06 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:06 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:57, 58, and 59.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:57” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:57, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:57, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:57, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:58” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:58, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:58, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:58, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:59” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:59, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:59, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:59, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:60 (or a variant of SEQ ID NO:60 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:61 (or a variant of SEQ ID NO:61 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:62 (or a variant of SEQ ID NO:62 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:07. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:07. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:07.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:07, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:60, 61, and 62. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99/a sequence identity to the amino acid sequence set forth in SEQ ID NO:07, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:60, 61, and 62.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:07 or the amino acid set forth in SEQ ID NO:07 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:07 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:60, 61, and 62.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:60” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:60, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:60, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:60, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:61” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:61, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:61, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:61, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:62” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:62, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:62, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:62, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

In another embodiment, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:63 (or a variant of SEQ ID NO:63 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:64 (or a variant of SEQ ID NO:64 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:65 (or a variant of SEQ ID NO:65 with one or two amino acid modifications). An example of such an antibody domain having these CDRs and the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) includes, without limitation, the VH domain set forth in TABLE 3.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:08. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:08. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include (a) a heavy chain variable domain that includes an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:08.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:08, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:63, 64, and 65. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein can include a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:08, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:63, 64, and 65.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:08 or the amino acid set forth in SEQ ID NO:08 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:08 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:63, 64, and 65.

As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:63” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:63, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:63, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:63, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:64” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:64, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:64, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:64, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:65” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:65, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:65, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:65, provided that the binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) maintains its basic ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide).

As indicated herein, the amino acid sequences described herein can include amino acid modifications (e.g., the articulated number of amino acid modifications). Such amino acid modifications can include, without limitation, amino acid substitutions, amino acid deletions, amino acid additions, and combinations. In some cases, an amino acid modification can be made to improve the binding and/or contact with an antigen and/or to improve a functional activity of a binder (e.g., an antibody, antigen binding fragment, antibody domain, a CAR, a cell engager, and/or an ADC) provided herein. In some cases, an amino acid substitution within an articulated sequence identifier can be a conservative amino acid substitution. For example, conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In some cases, an amino acid substitution within an articulated sequence identifier can be a non-conservative amino acid substitution. Non-conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, without limitation, substituting (a) a hydrophilic residue (e.g., serine or threonine) for a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) a cysteine or proline for any other residue; (c) a residue having a basic side chain (e.g., lysine, arginine, or histidine) for a residue having an acidic side chain (e.g., aspartic acid or glutamic acid); and (d) a residue having a bulky side chain (e.g., phenylalanine) for glycine or other residue having a small side chain.

Methods for generating an amino acid sequence variant (e.g., an amino acid sequence that includes one or more modifications with respect to an articulated sequence identifier) can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of a nucleic acid encoding the antibody or fragment thereof. See, for example, Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially-derivatized amino acids) can be used to generate an amino acid sequence variant provided herein.

When designing a single chain antibody (e.g., a scFv) having a heavy chain variable domain and a light chain variable domain, the two regions can be directly connected or can be connected using any appropriate linker sequence. For example, a heavy chain variable domain having the CDRs of TABLE 4 can be directly connected to a light chain variable domain via a linker sequence. Examples of linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain to create a scFv include, without limitation, those linkers set forth in FIG. 9 or FIG. 10.

The binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, and/or ADCs) provided herein can be produced using any appropriate method. For example, the binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, and/or cell engagers) provided herein can be produced in recombinant host cells. For example, a nucleic acid encoding a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein can be constructed, introduced into an expression vector, and expressed in suitable host cells. TABLE 6 includes examples of nucleic acid sequences that encode exemplary binders (e.g., antibody domains) described herein. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein can be recombinantly produced in prokaryotic hosts such as E. coli, Bacillus brews, Bacillus subtilis, Bacillus megateriurn, Lactobacillus zeae/casei, or Lactobacillus paracasei. A binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein also can be recombinantly produced in eukaryotic hosts such as yeast (e.g., Pichia pastoris. Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), filamentous fungi of the genera Trichoderma (e.g., T. reesei) and Aspergillus (e.g., A. niger and A. oryzae), protozoa such as Leishmania tarentolae, insect cells, or mammalian cells (e.g., mammalian cell lines such as Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, or human embryonic kidney cell line HEK293). See, for example, the Frenzel et al. reference (Front Immunol., 4:217 (2013)).

In some cases, an antigen binding fragment or antibody domain provided herein can be produced by proteolytic digestion of an intact antibody. For example, an antigen binding fragment can be obtained by treating an antibody with an enzyme such as papain or pepsin. Papain digestion of whole antibodies can be used to produce F(ab)₂or Fab fragments, while pepsin digestion of whole antibodies can be used to produce F(ab′)₂or Fab′ fragments.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be substantially pure. The term “substantially pure” as used herein with reference to a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) refers to the binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) as being substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein is any binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) that is removed from its natural environment and is at least 60% pure. A substantially pure binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be at least about 65, 70, 75, 80, 85, 90, 95, or 99% pure.

This document also provides bispecific binders (e.g., bispecific antibodies, bispecific antigen binding fragments, and/or bispecific antibody domains) that bind to two different epitopes with at least one being an epitope of an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). In some cases, a bispecific binder provided herein can be designed to bind to two different epitopes of the same FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). In some cases, a bispecific binder provided herein can bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and to an epitope on a different polypeptide (e.g., a CD3 polypeptide). Bispecific binders can be produced by chemically conjugating two different binders (e.g., antibodies, antigen binding fragments, and/or antibody domains) together. Bispecific binders also can be produced by fusing two antibody-producing cells, e.g., hybridomas, to make a hybrid cell line that produces two different heavy and two different light chains within the same cell, which can result in, for example, bispecific IgG molecules. See, Brinkmann and Kontermann, MAbs., 9(2):182-212 (2017).

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein can be fused or conjugated (e.g., covalently or non-covalently attached) to another polypeptide or other moiety to provide a fusion protein or conjugate. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein can be conjugated (e.g., covalently or non-covalently attached) to a polymer (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), and/or polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, a fluorescent substance, a luminescent substance, a hapten, an enzyme, a metal chelate, a drug, a radioisotope, and/or a cytotoxic agent. Any appropriate method can be used to conjugate (e.g., covalently or non-covalently attach) another polypeptide or other moiety to a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein. For example, another polypeptide or other moiety can be conjugated to a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein using the methods described in U.S. Pat. No. 8,021,661.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be modified with a moiety that improves its stabilization and/or retention in circulation, for example, in blood, serum, or other tissues by, for example, at least 1.5-, 2-, 5-, 10-, or 50-fold. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be attached (e.g., covalently or non-covalently attached) to a polymer such as a substantially non-antigenic polymer. Examples of substantially non-antigenic polymers that can be used as described herein include, without limitation, polyalkylene oxides and polyethylene oxides. In some cases, a polymer used herein can have any appropriate molecule weight. For example, a polymer having an average molecular weight from about 200 Daltons to about 35,000 Daltons (e.g., from about 1,000 to about 15,000 Daltons or from about 2,000 to about 12,500 Daltons) can be used. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be attached (e.g., covalently or non-covalently) to a water-soluble polymer. Examples of water-soluble polymers that can be used as described herein include, without limitation, hydrophilic polyvinyl polymers, polyvinylalcohol, polyvinylpyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycols, polyoxyethylenated polyols, and copolymers thereof and/or block copolymers thereof provided that the water solubility of the copolymer or block copolymers is maintained.

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be attached (e.g., covalently or non-covalently attached) to one or more polyoxyalkylenes (e.g., polyoxyethylene, polyoxypropylene, or block copolymers of polyoxyethylene and polyoxypropylene), polymethacrylates, carbomers, branched or unbranched polysaccharides, or combinations thereof. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein can be covalently attached to polyoxyethylene.

This document also provides ADCs. The term “ADC” as used herein refers to a conjugate that includes (a) an antigen binding domain and (b) at least one drug covalently linked directly or indirectly to that antigen binding domain. In some cases, an ADC described herein can include (a) an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) and (b) at least one drug covalently linked directly or indirectly to that antigen binding domain. Any appropriate binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein and having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can be used as an antigen binding domain to make an ADC described herein. For example, any of the binders set forth in TABLE 3 can be used to make an ADC having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). Examples of drugs that can be used to make an ADC described herein include, without limitation, auristatins (e.g., monomethyl auristatin E (MMAE)), mertansine (DM-1), and pyrrolobenzodiazepine (PBD) dimers. Any appropriate ADC linker can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) to form an ADC provided herein. For example, cleavable or non-cleavable ADC linkers can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) to form an ADC provided herein. Examples of ADC linkers can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) to form an ADC provided herein include, without limitation, ADC disulfide linkers, ADC hydrazone linkers, ADC peptide linkers, ADC thioether linkers, and ADC PEG-containing linkers.

This document also provides nucleic acid molecules (e.g., isolated nucleic acid molecules) having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein. For example, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence encoding a VH domain set forth in TABLE 3. A nucleic acid provided herein (e.g., an isolated nucleic acid molecule) can be single stranded or double stranded nucleic acid of any appropriate type (e.g., DNA, RNA, or DNA/RNA hybrids).

This document also provides vectors (e.g., plasmid vectors or viral vectors) containing one or more nucleic acids provided herein. An example of a plasmid vector that can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein includes, without limitation, phagemids. Examples of viral vectors that can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein include, without limitation, retroviral vectors, parvovirus-based vectors (e.g., adenoviral-based vectors and adeno-associated virus (AAV)-based vectors), lentiviral vectors (e.g., herpes simplex (HSV)-based vectors), poxviral vectors (e.g., vaccinia virus-based vectors and fowlpox virus-based vectors), and hybrid or chimeric viral vectors. For example, a viral vector having an adenoviral backbone with lentiviral components such as those described elsewhere (Zheng et al., Nat. Biotech., 18(2): 176-80 (2000); WO 98/22143; WO 98/46778; and WO 00/17376) or viral vectors having an adenoviral backbone with AAV components such as those described elsewhere (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996)) can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein.

In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding scFv or antibody domain (e.g., a VH domain) provided herein. In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding CAR provided herein. In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding cell engager provided herein.

A vector provided herein (e.g., a plasmid vector or viral vector provided herein) can include any appropriate promoter and other regulatory sequence (e.g., transcription and translation initiation and termination codons) operably linked the nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein. In some cases, a promoter used to drive expression can be a constitutive promotor or a regulatable promotor. Examples of regulatable promoters that can be used as described herein include, without limitation, inducible promotors, repressible promotors, and tissue-specific promoters. Examples of viral promotors that can be used as described herein include, without limitation, adenoviral promotors, vaccinia virus promotors, CMV promotors (e.g., immediate early CMV promotors) and AAV promoters.

Any appropriate method can be used to make a nucleic acid molecule (or vector such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein. For example, molecule cloning techniques can be used to make a nucleic acid molecule (or vector such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, N Y (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).

This document also provides host cells that include a nucleic acid provided herein (e.g., a nucleic acid having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein). Host cells that can be designed to include one or more nucleic acids provided herein can be prokaryotic cells or eukaryotic cells. Examples of prokaryotic cells that can be designed to include a nucleic acid provided herein include, without limitation, E. coli(e.g., Tb-1, TG-1, DH5α, XL-Blue MRF (Stratagene), SA2821, or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens, or Pseudomonas (e.g., P. aeruginosa) cells. Examples of eukaryotic cells that can be designed to include a nucleic acid provided herein include, without limitation, insect cells (e.g., Sf9 or Ea4 cells), yeast cells (e.g., S. cerevisiae cells), and mammalian cells (e.g., mouse, rat, hamster, monkey, or human cells). For example, VERO cells, HeLa cells, 3T3 cells, chinese hamster ovary (CHO) cells, W138 BHK cells, COS-7 cells, and MDCK cells can be designed to include a nucleic acid provided herein. Any appropriate method can be used to introduce one or more nucleic acids provided herein (e.g., a vector such as a plasmid vector or viral vector having a nucleic acid sequence encoding at least part of a binder provided herein) into a host cell. For example, calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, electroporation, or combinations thereof can be used to introduce a nucleic acid provided herein into a host cell (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, N Y (1989); Davis et al., Basic Methods in Molecular Biology (1986); and Neumann et al., EMBO J., 1:841 (1982)).

In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be designed to express one or more nucleic acids encoding a CAR described herein. For example, a population of T cells can be infected with viral vectors designed to express nucleic acid encoding a CAR described herein (e.g., a CAR having the ability to bind to an FGFR4 polypeptide).

In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be designed to express one or more nucleic acids encoding a cell engager described herein. For example, a population of T cells can be infected with viral vectors designed to express nucleic acid encoding a cell engager described herein (e.g., a cell engager having the ability to bind to an FGFR4 polypeptide).

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein can be produced using a method that includes (a) introducing nucleic acid encoding the polypeptide into a host cell; (b) culturing the host cell in culture medium under conditions sufficient to express the polypeptide; (c) harvesting the polypeptide from the cell or culture medium; and (d) purifying the polypeptide (e.g., to reach at least 50, 60, 70, 80, 90, 95, 97, 98, or 99% purity).

In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein, a nucleic acid provided herein (e.g., nucleic acid encoding an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein), a vector provided herein (e.g., a viral vector designed to express an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein), and/or a host cell provided herein (e.g., a host cell designed to express an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein) can be formulated as a pharmaceutical composition for administration to a mammal (e.g. a human) having a cancer including but not limited to rhabdomyosarcomas, as a therapy for that mammal. In some cases, a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein, a nucleic acid provided herein (e.g., nucleic acid encoding an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein), a vector provided herein (e.g., a viral vector designed to express an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein), and/or a host cell provided herein (e.g., a host cell designed to express an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager provided herein) can be formulated as a pharmaceutical composition for administration to a mammal (e.g. a human) to reduce the number of cancer cells within the mammal and/or to increase the survival of the mammal suffering from cancer. For example, a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein having the ability to bind to an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) can be formulated as a pharmaceutical composition for administration to a mammal (e.g. a human). In some cases, a pharmaceutical composition provided herein can include a pharmaceutically acceptable carrier such as a buffer, a salt, a surfactant, a sugar, a tonicity modifier, or combinations thereof as, for example, described elsewhere (Gervasi, et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharmaceutically acceptable carriers that can be used to make a pharmaceutical composition provided herein include, without limitation, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, a sugar (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or combinations thereof. For example, a pharmaceutical composition designed to include a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, cell engager, and/or ADC) provided herein (or a nucleic acid, a vector, or a host cell provided herein) can be formulated to include a buffer (e.g., an acetate, citrate, histidine, succinate, phosphate, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar such as sucrose. Other ingredients that can be included within a pharmaceutical composition provided herein include, without limitation, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA), anticancer agents such as enzalutamide, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosine, bortezomib, carfilzomib, batimastat, ganetespib, obatoclax, navitoclax, taxol, paclitaxel, or bevacizumab, or combinations thereof. For example, a pharmaceutical composition provided herein can be formulated to include one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cells designed to express a CAR having the ability to bind to an FGFR4 polypeptide, one or more cell engagers, and/or one or more ADCs) provided herein in combination with one or more checkpoint inhibitors such as anti-PD-1 antibodies or PD-1 inhibitors (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, or AMP-514), anti-PD-L1 antibodies or PD-L1 inhibitors (e.g., avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189), and/or anti-CTLA-4 antibodies (e.g., ipilimumab).

In some cases, when a pharmaceutical composition is formulated to include one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cells designed to express a CAR having the ability to bind to an FGFR4 polypeptide, one or more cell engagers, and/or one or more ADCs) provided herein, any appropriate concentration of the binder can be used. For example, a pharmaceutical composition provided herein can be formulated to be a liquid that includes from about 1 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 2 mg to about 200 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR⁺ cell population, cell engager, and/or ADC) provided herein per mL. In another example, a pharmaceutical composition provided herein can be formulated to be a solid or semi-solid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein. In some cases, a pharmaceutical composition containing a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be formulated as a dosage form with a titer of the binder being from about 1×10⁵to about 1×10¹²(e.g., from about 1×10⁵to about 1×10¹⁰, from about 1×10⁵to about 1×10⁸, from about 1×10⁶to about 1×10¹², from about 1×10⁶to about 1×10¹², from about 1×10⁸to about 1×10¹², from about 1×10⁹to about 1×10¹², from about 1×10⁶to about 1×10¹¹, or from about 1×10⁷to about 1×10¹⁰).

In some cases, when a pharmaceutical composition is formulated to include one or more nucleic acids (e.g., vectors such as viral vectors) encoding at least part of a binder (e.g., an antibody, antigen binding fragment, antibody domain, CAR, and/or cell engager) provided herein, any appropriate concentration of the nucleic acid can be used. For example, a pharmaceutical composition provided herein can be formulated to be a liquid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 2 mg to about 200 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a nucleic acid provided herein per mL. In another example, a pharmaceutical composition provided herein can be formulated to be a solid or semi-solid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of a nucleic acid provided herein.

In some cases, a pharmaceutical composition designed to include a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein can be formulated to include one or more agents capable of reducing aggregation of the binder when formulated. Examples of such agents that can be used as described herein include, without limitation, methionine, arginine, lysine, aspartic acid, glycine, or glutamic acid, and combinations thereof. In some cases, one or more of these amino acids can be included within the formulation at a concentration from about 0.5 mM to about 145 mM (e.g., from about 1 mM to about 145 mM, from about 10 mM to about 145 mM, from about 100 mM to about 145 mM, from about 0.5 mM to about 125 mM, from about 0.5 mM to about 100 mM, from about 0.5 mM to about 75 mM, or from about 10 mM to about 100 mM).

A pharmaceutical composition provided herein can be in any appropriate form. For example, a pharmaceutical composition provided herein can designed to be a liquid, a semi-solid, or a solid. In some cases, a pharmaceutical composition provided herein can be a liquid solution (e.g., an injectable and/or infusible solution), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository. In some cases, a pharmaceutical composition provided herein can be lyophilized. In some cases, a pharmaceutical composition provided herein (e.g., a pharmaceutical composition that includes one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein can be formulated with a carrier or coating designed to protect against rapid release. For example, a pharmaceutical composition provided herein can be formulated as a controlled release formulation or as a regulated release formulation as described elsewhere (U.S. Patent Application Publication Nos. 2019/0241667; 2019/0233522; and 2019/0233498, all of which are expressly incorporated by reference in their entireties).

This document also provides methods for administering a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) to a mammal (e.g., a human). For example, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, and/or host cell (e.g., CAR cells) provided herein) can be administered to a mammal (e.g., a human) having cancer including but not limited to rhabdomyosarcomas, as a therapy for that mammal. In some cases, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, and/or host cell (e.g., CAR⁺ cells) provided herein) can be administered to a mammal (e.g. a human) to reduce the number of cancer cells within the mammal and/or to increase the survival of the mammal suffering from a cancer, including but not limited to rhabdomyosarcomas.

Any appropriate cancer can be treated or inhibited using a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein). For example, a mammal (e.g., a human) having cancer, including but not limited to rhabdomyosarcomas, can be treated or inhibited by administering a composition (e.g., a pharmaceutical composition) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein to that mammal. Examples of cancers that can be treated or inhibited as described herein include, without limitation, Ewing's sarcoma, acute myeloid leukemia (AML), liver cancer, colorectal cancer, brain cancer, skin cancer (e.g., melanoma), lung cancer, prostate cancer, breast cancer (e.g., PR positive breast cancer, ER positive breast cancer, HER2 positive breast cancer, or triple negative breast cancer), ovarian cancer, cervical cancer, esophageal cancer, glioma, kidney cancer, mesothelioma, and pancreatic cancer. In some cases, a solid cancer such as liver cancer, colorectal cancer, brain cancer, skin cancer (e.g., melanoma), lung cancer, prostate cancer, breast cancer (e.g., PR positive breast cancer, ER positive breast cancer, HER2 positive breast cancer, or triple negative breast cancer), ovarian cancer, cervical cancer, esophageal cancer, glioma, kidney cancer, mesothelioma, or pancreatic cancer can be treated as described herein. In some cases, cancer such as lymphoma (e.g., B cell lymphomas such as diffuse large cell lymphoma (DLBCL)), leukemia (e.g., chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL)), or acute myeloid leukemia can be treated as described herein. In some cases, a mammal (e.g., a human) having an FGFR4 cancer (e.g., an FGFR4⁺ Ewing's sarcoma, an FGFR4⁺ AML, an FGFR4⁺ liver cancer, FGFR4⁺ colorectal cancer, FGFR4⁺ brain cancer, FGFR4⁺ skin cancer (e.g., FGFR4⁺ melanoma), FGFR4⁺ lung cancer, FGFR4⁺ prostate cancer, FGFR4⁺ breast cancer (e.g., FGFR4⁺ PR positive breast cancer, FGFR4 ER positive breast cancer, FGFR4⁺ HER2 positive breast cancer, or FGFR4⁺ triple negative breast cancer), FGFR4⁺ ovarian cancer, FGFR4⁺ cervical cancer, FGFR4⁺ esophageal cancer, FGFR4⁺ glioma, FGFR4⁺ kidney cancer, FGFR4⁺ mesothelioma, or FGFR4⁺ pancreatic cancer) can be administered a composition (e.g., a pharmaceutical composition) containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein to treat that mammal or inhibit the cancer (e.g., to reduce the number of cancer cells within the mammal).

Any appropriate method can be used to administer a composition (e.g., a pharmaceutical composition) provided herein to a mammal (e.g., a human). For example, a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein such as one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs provided herein) can be administered to a mammal (e.g., a human) intravenously (e.g., via an intravenous injection or infusion), subcutaneously (e.g., via a subcutaneous injection), intraperitoneally (e.g., via an intraperitoneal injection), orally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some cases, the route and/or mode of administration of a composition (e.g., a pharmaceutical composition provided herein) can be adjusted for the mammal being treated.

In some cases, an effective amount of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be an amount that reduces the number of cancer cells within a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective amount of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be an amount that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition. For example, an effective amount of a binder (e.g., an antibody, antigen binding fragment, antibody domain, cell engager, and/or ADC) provided herein can be from about 0.001 mg/kg to about 100 mg/kg (e.g., from about 0.001 mg/kg to about 90 mg/kg, from about 0.001 mg/kg to about 80 mg/kg, from about 0.001 mg/kg to about 70 mg/kg, from about 0.001 mg/kg to about 60 mg/kg, from about 0.001 mg/kg to about 50 mg/kg, from about 0.001 mg/kg to about 40 mg/kg, from about 0.001 mg/kg to about 30 mg/kg, from about 0.005 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.5 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 100 mg/kg, from about 5 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 30 mg/kg, from about 0.15 mg/kg to about 25 mg/kg, from about 0.2 mg/kg to about 20 mg/kg, from about 0.5 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 2 mg/kg to about 20 mg/kg, from about 5 mg/kg to about 30 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 15 mg/kg to about 30 mg/kg, from about 20 mg/kg to about 30 mg/kg, from about 3 mg/kg to about 30 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, or from about 1 mg/kg to about 3 mg/kg). The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the severity of cancer when treating a mammal having cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective amount of a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein) that is administered.

In some cases, an effective frequency of administration of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a frequency that reduces the number of cancer cells within a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective frequency of administration of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a frequency that increases the survival time of a mammal having a cancer, including but not limited to rhabdomyosarcomas, as compared to a control mammal having a comparable cancer and not administered the composition. For example, an effective frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be from about twice daily to about once a year (e.g., from about twice daily to about once a month, from about twice daily to about once a week, from about once daily to about once a month, or from one once daily to about once a week). In some cases, the frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be daily. The frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective frequency of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein).

In some cases, an effective duration of administration of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a duration that reduces the number of cancer cells within a mammal without producing significant toxicity to the mammal. In some cases, an effective duration of administration of a composition containing one or more binders (e.g., one or more antibodies, one or more antigen binding fragments, one or more antibody domains, one or more cell engagers, and/or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR⁺ cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a duration that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition. For example, an effective duration of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can vary from a single time point of administration to several weeks to several months (e.g., 4 to 12 weeks). Multiple factors can influence the actual effective duration used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective duration of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein).

In some cases, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be used to detect the presence or absence of an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) in vitro, in situ, or in vivo (e.g., in vivo imaging within a mammal such as a human). For example, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be designed to include a label (e.g., a covalently attached radioactive, enzymatic, colorimetric, or fluorescent label). The labelled binder can be used to detect the presence or absence of an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) within a biological sample in vitro. Examples of biological samples that can be assessed using a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein include, without limitation, serum samples, plasma samples, tissue samples, biopsy samples, cell line samples, and tissue culture samples. In some cases, a biological sample that can be assessed as described herein can include mammalian body tissues and/or cells such as leukocytes, ovary tissue or cells, prostate tissue or cells, heart tissue or cells, placenta tissue or cells, pancreas tissue or cells, liver tissue or cells, spleen tissue or cells, lung tissue or cells, breast tissue or cells, head and neck tissue or cells, endometrium tissue or cells, colon tissue or cells, colorectal tissue or cells, cervix tissue or cells, stomach tissue or cells, or umbilical tissue or cells that may express an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide). In some cases, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be immobilized, e.g., on a support, and retention of an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) from a biological sample on the support can be detected, and/or vice versa. In some cases, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein can be used in applications such as fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and/or cell sorting (e.g., fluorescence activated cell sorting).

In some cases, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein containing a label (e.g., a covalently attached radioactive label) can be used to detect the presence or absence of an FGFR4 polypeptide (e.g., a human FGFR4 polypeptide) within a mammal (e.g., a human). For example, a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein that is labelled (e.g., covalently labelled) with a radiolabel or an MRI detectable label can be administered to a mammal (e.g., a human), and that mammal can be assessed by detecting the detectable label. In some cases, a mammal can be scanned to evaluate the location(s) of a labelled binder provided herein within the mammal. For example, the mammal can be imaged using NMR or other tomographic techniques.

Examples of labels that can be attached (e.g., covalently or non-covalently attached) to a binder (e.g., an antibody, antigen binding fragment, and/or antibody domain) provided herein include, without limitation, radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ^99mTc, ³²P, ³³P, ¹²⁵I, ³H, ¹⁴C, or ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, or enzymatic markers such as a peroxidase or a phosphatase. In some cases, short-range radiation emitters such as isotopes detectable by short-range detector probes can be used.

Some embodiments of the methods and compositions provided herein include chimeric antigen receptors (CARs), which specifically bind to fibroblast growth factor receptor 4 (FGFR4). Some embodiments include nucleic acids encoding such CARs, and cells containing such CARs. Some embodiments include the use of such CARs therapies for a cancer, such as an FGFR4-expressing cancer, such as a rhabdomyosarcoma.

Genomics can be used to design CARs for CAR T-cell immunotherapy (Orentas R J, et al., 2012, Front Oncol 2:194). Affymetrix-based expression analysis of ARMS and ERMS patient samples, comparing tumor to normal gene expression demonstrated that even though a number of transcripts were shared between disease types, fusion positive (ARMS) samples alone identified FGFR4 as a favorable target. TABLE 1 lists transcripts overexpressed in ARMS in comparison to normal tissue and shows the top 12 transcripts ranked by difference from averaged normal tissue by T statistic. The broad distribution of MCAM/CD146 and the potential toxicity of targeting CHRNA1 makes FGFR4 a target.

TABLE 1

Rank
Gene ID
T value
P value

1
CD146/MCAM*
27.68
2.8 × 10E−20
Melanoma cell adhesion molecule (MUC18)

2
CHRNA1*
24.21
7.2 × 10E−19
Cholinergic R, nicotinic, alpha 1 subunit (muscle)

3
CD334/FGFR4*
19.67
1.0 × 10E−16
Fibroblast growth factor receptor 4

4
MEGF10*
19.19
1.8 × 10E−16
Multiple EGF-like-domains 10

5
ACVR2A*
14.78
7.3 × 10E−14
Activin A receptor, type IIA

6
KCNN3
14.00
2.5 × 10E−13
K+, Ca-activ. channel subfam N, 3

7
CDH15/MCAD
13.85
3.1 × 10E−13
Cadherin 15, M-cadherin (myotubule)

8
SEMA6B*
12.15
5.5 × 10E−12
Semaphorin 6B

9
GPC2*
11.89
8.7 × 10E−12
Glypican 2 (cerebroglycan)

10
CD271/NGFR
11.43
2.0 × 10E−11
NGFR, nerve growth factor receptor

11
CACNA1I
11.30
2.6 × 10E−11
Ca channel, voltage-dep., alpha1 subunit

12
MET
11.07
4.0 × 10E−11
Met proto-oncogene

*Transcripts shared by both ARMS and ERMS.

FGFR4 is the highest ranked target that can be safely approached in ARMS, and is not expressed in ERMS.

Studies have shown a role of FGFR4 in RMS tumor biology demonstrating that FGFR4 expression is associated with advanced stage RMS and poor survival, and that FGFR4 knockdown in a human RMS cell line led to decreased pathogenicity in a mouse model of disease (Taylor J G el al., 2009, J Clin Invest 119-3395-3407). A mutated tyrosine kinase receptor in RMS has been identified. In 7 of 94 primary RMS tumors, FGFR4 tyrosine kinase mutations were identified, which served to increase autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential. The function of FGFR4 as an oncogenic driver has been documented, for example, a mouse model expressing the common FGFR4^V550Emutation was used to demonstrate that the dual PI3K/mTOR inhibitor omipalisib was able to impact tumor progression by blocking these down-stream FGFR4 signals (McKinnon T, et al., 2018, Oncogene 37:2630-2644). Other studies have shown that FGFR4 overexpression leads to resistance to chemotherapy and the induction of an anti-apoptotic state (Roidl A., et al., 2009, Clin Cancer Res 15:2058-2066).

GD2-specific CARs were developed for the treatment of pediatric solid tumors (Long A H, et al., 2016, Cancer Immunol Res 4:869-880). In looking at GD2 expression, all osteosarcomas (n=18), 13% of RMS (ARMS, n=14, ERMS, n=1), and 20% of Ewing sarcomas (n-=35), were positive. The ability of a GD2-CAR to control tumor xenografts in vivo was tested. Although GD2-CARs, especially in the 4-1BB-zeta chain format, were effective against a Kelly neuroblastoma cell line in vivo in NSG mice, GD2-CARs were ineffective against the 143b osteosarcoma cell line. Despite this ineffectiveness, and low incidence on RMS, these studies established that the GD2-CAR self-associated on the T cell surface leading to target cell-independent metabolic exhaustion, and that this was partially rescued by the inclusion of a CD137/4-1BB signaling domain in the CAR construct (Long A H, et al., 2015, Nat Med 21:581-590). This optimized 4-1BBz CAR was still poorly effective, unless mice were also treated with all-trans retinoic acid (ATRA). ATRA inhibited the accumulation of monocytic myeloid derived suppressor cells (mMDSCs) in the tumor lesion and diminished their inhibitory effect on T cell proliferation.

The cell surface protein FGFR4 is expressed during development, however expression on normal tissue is low to non-existent in mature tissues. Nevertheless, FGFR4 is highly overexpressed in certain human tumors, including rhabdomyosarcoma. Disclosed herein are a series of binding moieties identified through screening methods and used to generate polynucleotides encoding CARs.

Certain embodiments of the methods and compositions provided herein can include aspects disclosed in Lang L, et al., 2019, Cells 8:3.1; Chen C, et al., 2005, Hybridoma, 24(3):152-159; U.S. Pat. Nos. 9,266,955; 8,394,927; 10,358,498; and 11,078,286, which are each expressly incorporated by reference in its entirety.

Certain Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

As used herein, “a” or “an” may mean one or more than one.

“About” as used herein when referring to a measurable value is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value.

As used herein, “nucleic acid” or “nucleic acid molecule” have their plain and ordinary meaning in view of the whole specification and may to refer to, for example, polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), or fragments generated by any of ligation, scission, endonuclease action, or exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA or RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, or azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars or carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, or phosphoramidate, and the like. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. In some embodiments, a nucleic acid sequence encoding a fusion protein is provided. In some embodiments, the nucleic acid encoding the chimeric antigen receptor specific for FGFR4.

As used herein, “coding for” or “encoding” has its plain and ordinary meaning when read in light of the specification, and includes, for example, the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.

As used herein, “chimeric antigen receptor” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a synthetically designed receptor comprising a ligand binding domain of an antibody or other protein sequence that binds to a molecule associated with a disease or disorder and is, preferably, linked via a spacer domain to one or more intracellular signaling domains of a cell, such as a T cell, or other receptors, such as one or more costimulatory domains. Chimeric receptor can also be referred to as artificial cell receptors or T cell receptors, chimeric cell receptors or T cell receptors, chimeric immunoreceptors, or CARs. These receptors can be used to graft the specificity of a monoclonal antibody or binding fragment thereof onto a cell, preferably a T-cell, with transfer of their coding sequence facilitated by viral vectors, such as a retroviral vector or a lentiviral vector. CARs can be, in some instances, genetically engineered T cell receptors designed to redirect T cells to target cells that express specific cell-surface antigens. T cells can be removed from a subject and modified so that they can express receptors that can be specific for an antigen by a process called adoptive cell transfer. The T cells are reintroduced into the patient where they can then recognize and target an antigen. CARs are also engineered receptors that can graft an arbitrary specificity onto an immune receptor cell. CARs are considered by some investigators to include the antibody or antibody fragment, preferably an antigen binding fragment of an antibody, the spacer, signaling domain, and transmembrane region. Due to the surprising effects of modifying the different components or domains of the CAR described herein, such as the epitope binding region (for example, antibody fragment, scFv, or portion thereof), spacer, transmembrane domain, and/or signaling domain), the components of the CAR are frequently distinguished throughout this disclosure in terms of independent elements. The variation of the different elements of the CAR can, for example, lead to stronger binding affinity for a specific epitope or antigen.

The CARs graft the specificity of a monoclonal antibody or binding fragment thereof or scFv onto a T cell, with the transfer of their coding sequence facilitated by vectors. In order to use CARs as a therapy for a subject in need, a technique called adoptive cell transfer is used in which T cells are removed from a subject and modified so that they can express the CARs that are specific for an antigen. The T cells, which can then recognize and target an antigen, are reintroduced into the patient.

As used herein, an “antibody” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a large Y-shape protein produced by plasma cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody protein can comprise four polypeptide chains, two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains can contain about 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids or any number of amino acids in between in a range defined by any two of these values and are classified into different categories according to their size and function. In some embodiments, the ligand binding domain comprises a CDR, an antibody or binding fragment thereof or scFv, a receptor ligand or mutants thereof, peptide, and/or polypeptide affinity molecule or binding partner. In some embodiments, the ligand binding domain is an antibody fragment, desirably, a binding portion thereof. In some embodiments, the antibody fragment or binding portion thereof present on a CAR is specific for a ligand on a B-cell. In some embodiments, the antibody fragment or binding portion thereof present on a CAR or TcR is specific for a ligand. In some embodiments, the antibody fragment or binding portion thereof present on a CAR is specific for FGFR4. In some embodiments, the ligand binding domain is an antibody fragment or a binding portion thereof, such as a VH domain, or a single chain variable fragment (scFv) containing VH and VL domains. In some embodiments, the antibody fragment or binding portion thereof present on a CAR comprises one or more domains from a humanized antibody, or binding portion thereof.

As used herein, “complementarity-determining regions” or “CDR” includes certain portions of the variable region in an antibody or antigen binding fragment thereof, which forms the binding and specificity of various specific antibodies to their particular antigen. Variability is not uniformly distributed throughout an antibody variable region, and may be focused concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chain. The more conserved part of the variable region is called the framework region (FR). The variable regions of the natural heavy and light chains each contain four FR regions, which are substantially in a β-folded configuration, joined by three CDRs which form a linking loop, and in some cases can form a partially s-folded structure. The CDRs in each chain are closely adjacent to the others by the FR regions and form an antigen-binding site of the antibody with the CDRs of the other chain. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effects or functions, for example, involving in antibody-dependent cytotoxicity of the antibodies. In some embodiments, a CDR can be found within antibody region as numbered by Kabat as follows: for the light chain, CDR1 at amino acids 24-34; CDR2 at amino acids 50-56; CDR3 at amino acids 89-97; for the heavy chain, CDR1 at amino acids 31-35; CDR2 at amino acids 50-65; and CDR3 at amino acids 95-102. CDR regions in antibodies and antigen fragments thereof, such as scFVs, can be readily determined.

As used herein, a “single chain variable fragment” or “scFv” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to 25 amino acids or about 25 amino acids. In some embodiments, a CAR is provided, wherein the CAR comprises a ScFv or a VH domain specific for FGFR4.

The strength of binding of a ligand is referred to as the binding affinity and can be determined by direct interactions and solvent effects. A ligand can be bound by a “ligand binding domain.” A ligand binding domain, for example, can refer to a conserved sequence in a structure that can bind a specific ligand or a specific epitope on a protein. The ligand binding domain or ligand binding portion can comprise an antibody or binding fragment thereof or scFv, a VH domain, a receptor ligand or mutants thereof, peptide, and/or polypeptide affinity molecule or binding partner. Without being limiting, a ligand binding domain can be a specific protein domain or an epitope on a protein that is specific for a ligand or ligands.

Some embodiments include a spacer. In some embodiments, the peptide spacer is 15 amino acids or less but not less than 1 or 2 amino acids. In some embodiments, the spacer is a polypeptide chain. In some aspects, the polypeptide chain may range in length, such as from 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240 amino acids or a length within a range defined by any two of the aforementioned lengths. A spacer can comprise any 20 amino acids, for example, in any order to create a desirable length of polypeptide chain in a CAR, which includes the amino acids arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine or tryptophan. A spacer sequence can be a linker between the ligand binding domain and the transmembrane domain of the CAR. In some embodiments, the chimeric antigen receptor further comprises a sequence encoding a spacer. In some embodiments, the spacer comprises a sequence with a length of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 or 240 amino acids or a length within a range defined by any two of the aforementioned lengths. In some embodiments, the spacer resides between the ligand binding domain and the transmembrane region of the CAR. In some embodiments, the spacer resides between the ligand binding domain of the CAR and the transmembrane region of the CAR.

A spacer may also be customized, selected, or optimized for a desired length so as to improve or modulate binding of the ligand binding domain to the target cell, which may increase cytotoxic efficacy. In some embodiments, the linker or spacer between the ligand binding domain and the transmembrane can be 25 to 55 amino acids in length (e.g., at least, equal to 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, 50, 51, 52, 53, 54, or 55 amino acids or a length within a range defined by any two of the aforementioned lengths).

In some embodiments, the spacer comprises a CD8 hinge. In some embodiments, the spacer comprises a hinge region of a human antibody. In some embodiments, the spacer comprises an IgG4 hinge. In some embodiments, the IgG4 hinge region is a modified IgG4 hinge. A “modified IgG4 hinge” as described herein can refer to a hinge region that can have at least 90%/6, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%/6, or 100% sequence identity or a sequence identity within a range defined by any two of the aforementioned percentages, with a hinge region amino acid sequence as set forth in a spacer, such as a spacer listed in TABLE 5. In some embodiments, the CAR comprises an S spacer, M spacer or an L spacer. Example sequences are listed in TABLE 5.

As used herein, a “de-immunized spacer” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a spacer that induces little to no immune response or a diminished or reduced immune response from a patient. In some embodiments, the CAR comprises a spacer, wherein the spacer does not induce an immune response in a subject, such as a human. It is important that the spacer does not induce an immune response or induces a reduced or diminished or low immune response in a subject, such as a human, in order to prevent or reduce the ability of the immune system to attack the chimeric antigen receptor.

In some embodiments, the transmembrane domain is a region of a membrane-spanning protein that is hydrophobic that can reside in the bilayer of a cell to anchor a protein that is embedded to the biological membrane. Without being limiting, the topology of the transmembrane domain can be a transmembrane alpha helix. In some embodiments, a CAR comprises a sequence encoding a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane sequence or a fragment thereof or a CD28 transmembrane sequence or a fragment thereof that is a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids or a length within a range defined by any two of the aforementioned lengths. In some embodiments, the CD8 transmembrane sequence or a fragment thereof or the CD28 transmembrane sequence or fragment thereof comprises 28 amino acids in length.

In some embodiments, the signaling domains, such as primary signaling domains or costimulatory domains, include an intracellular or cytoplasmic domain of a protein or a receptor protein that interacts with components within the interior of the cells and is capable of or configured to relay or participate in the relaying of a signal. Such interactions in some aspects can occur through the intracellular domain communicating via specific protein-protein or protein-ligand interactions with an effector molecule or an effector protein, which in turn can send the signal along a signal chain to its destination. In some embodiments, the signaling domain includes one or more co-stimulatory domains. In some embodiments, the one or more costimulatory domains include a signaling moiety that provides a T-cell with a signal, which, in addition to the primary signal provided by for instance the CD3 zeta chain of the TCR/CD3 complex, enhances a response such as a T-cell effector response, such as, for example, an immune response, activation, proliferation, differentiation, cytokine secretion, cytolytic activity, perforin or granzyme activity or any combination thereof. In some embodiments, the intracellular signaling domain or the co-stimulatory domain can include all or a portion of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3, or a ligand that specifically binds with CD83 or any combination thereof.

As used herein, a “ribosome skip sequence” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a sequence that during translation, forces the ribosome to “skip” the ribosome skip sequence and translate the region after the ribosome skip sequence without formation of a peptide bond. Several viruses, for example, have ribosome skip sequences that allow sequential translation of several proteins on a single nucleic acid without having the proteins linked via a peptide bond. As described herein, this is the “linker” sequence. In some embodiments of the nucleic acids provided herein, the nucleic acids comprise a ribosome skip sequence between the sequence for the chimeric antigen receptor and the sequence of the marker protein, such that the proteins are co-expressed and not linked by a peptide bond. In some embodiments, the ribosome skip sequence is a P2A, T2A, E2A or F2A sequence.

As used herein, a “marker sequence,” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a protein that is used for selecting or tracking a protein or cell that has a protein of interest. In the embodiments described herein, the fusion protein provided can comprise a marker sequence that can be selected in experiments, such as flow cytometry. In some embodiments, the marker is a truncated EGFR polypeptide (EGFRt), or a truncated HER2 polypeptide (HER2t).

As used herein, “signal sequence” for secretion, can also be referred to as a “signal peptide.” The signal peptide can be used for secretion efficiency and in some systems it is recognized by a signal recognition particle, which halts translation and directs the signal sequence to a SRP receptor for secretion. In some embodiments of the CARs provided herein, the CARs further comprise a signal sequence. In some embodiments, of the nucleic acid encoding a CAR, the nucleic acid comprises a sequence encoding a signal sequence. In some embodiments, the signal sequence is for targeting a protein to a cell membrane following translation of the protein.

As used herein, “vector” or “construct” has its plain and ordinary meaning when read in light of the specification, and includes, for example, a nucleic acid used to introduce heterologous nucleic acids into a cell that has regulatory elements to provide expression of the heterologous nucleic acids in the cell. Vectors include but are not limited to plasmid, minicircles, yeast, viral genomes, lentiviral vector, foamy viral vector, retroviral vector or gammaretroviral vector. The vector may be DNA or RNA, such as mRNA.

As used herein, “T-cells” or “T lymphocytes” can be from any mammal, preferably a primate, including monkeys or humans, a companion animal such as a dog, cat, or horse, or a domestic animal, such as a sheep, goat, or cattle. In some embodiments the T-cells are allogeneic (from the same species but different donor) as the recipient subject; in some embodiments the T-cells are autologous (the donor and the recipient are the same); in some embodiments the T-cells are syngeneic (the donor and the recipients are different but are identical twins).

As used herein, “T cell precursors” refer to lymphoid precursor cells that can migrate to the thymus and become T cell precursors, which do not express a T cell receptor. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classed as double-negative (CD4-CD8−) cells. As they progress through their development, they become double-positive thymocytes (CD4+ CD8+), and finally mature to single-positive (CD4+ CD8− or CD4-CD8+) thymocytes that are then released from the thymus to peripheral tissues.

As used herein, “hematopoietic stem cells” or “HSC” are precursor cells that can give rise to myeloid cells such as, for example, macrophages, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells and/or lymphoid lineages (such as, for example, T-cells, B-cells, or NK-cells). HSCs have a heterogeneous population in which three classes of stem cells exist, which are distinguished by their ratio of lymphoid to myeloid progeny in the blood (L/M).

As used herein, “CD4+ expressing T-cell,” or “CD4+ T-cell,” are used synonymously throughout, is also known as T helper cells, which play an important role in the immune system, and in the adaptive immune system. CD4+ T-cells also help the activity of other immune cells by releasing T-cell cytokines. These cells help, suppress or regulate immune responses. They are essential in B cell antibody class switching, in the activation and growth of cytotoxic T-cells, and in maximizing bactericidal activity of phagocytes, such as macrophages. CD4+ expressing T-cells have the ability to make some cytokines, however the amounts of cytokines made by CD4+ T-cells are not at a concentration that promotes, improves, contributes to, or induces engraftment fitness. As described herein, “CD4+ T-cells” are mature T helper-cells that play a role in the adaptive immune system.

As used herein, “CD8+ expressing T-cell” or “CD8+ T-cell,” are used synonymously throughout, is also known as a TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T-cell or killer T-cell. As described herein, CD8+ T-cells are T-lymphocytes that can kill cancer cells, virally infected cells, or damaged cells. CD8+ T-cells express T-cell receptors (TCRs) that can recognize a specific antigen. CD8+ T-cells express CD8 on the surface. CD8+ expressing T-cells have the ability to make some cytokines, however the amounts of cytokines made by CD8+ T-cells are not at a concentration that promotes, improves, contributes to, or induces engraftment fitness. “CD8 T-cells” or “killer T-cells” are T-lymphocytes that can kill cancer cells, cells that are infected with viruses or cells that are damaged.

Mature T cells express the surface protein CD4 and are referred to as CD4+ T-cells. CD4+ T-cells are generally treated as having a pre-defined role as helper T-cells within the immune system. For example, when an antigen-presenting cell expresses an antigen on MHC class II, a CD4+ cell will aid those cells through a combination of cell to cell interactions (e.g. CD40 and CD40L) and through cytokines. Nevertheless, there are rare exceptions; for example, sub-groups of regulatory T-cells, natural killer cells, and cytotoxic T-cells express CD4. All of the latter CD4+ expressing T-cell groups are not considered T helper cells.

As used herein, “central memory” T-cell (or “TCM”) refers to an antigen experienced CTL that expresses CD62L or CCR-7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA, as compared to naïve cells. In some embodiments, central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO, and/or CD95, and have decreased expression of CD54RA, as compared to naïve cells.

As used herein, “effector memory” T-cell (or “TEM”) refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells, and does not express or has decreased expression of CD45RA as compared to naïve cell. In some embodiments, effector memory cells are negative for expression of CD62L and/or CCR7, as compared to naïve cells or central memory cells, and have variable expression of CD28 and/or CD45RA.

As used herein, “naïve” T-cells refers to a non-antigen experienced T lymphocyte that expresses CD62L and/or CD45RA, and/or does not express CD45RO− as compared to central or effector memory cells. In some embodiments, naïve CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naïve T-cells including CD62L, CCR7, CD28, CD127, or CD45RA.

As used herein, “effector” “TE” T-cells refers to a antigen experienced cytotoxic T lymphocyte cells that do not express or have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme B or perforin or both, as compared to central memory or naïve T-cells.

As used herein, “cytokines” has its plain and ordinary meaning when read in light of the specification, and includes, for example, small proteins (5-25 kDa) that are important in cell signaling. Cytokines are released by cells and affect the behavior of other cells, and sometimes the releasing cell itself, such as a T-cell. Cytokines can include, for example, chemokines, interferons, interleukins, lymphokines, or tumor necrosis factor or any combination thereof. Cytokines can be produced by a broad range of cells, which can include, for example, immune cells like macrophages, B lymphocytes, T lymphocytes, mast cells, as well as, endothelial cells, fibroblasts, or various stromal cells.

Cytokines can act through receptors and are important in the immune system as the cytokines can modulate the balance between humoral and cell-based immune responses, and they can regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways. Without being limiting, cytokines can include, for example, Acylation stimulating protein, Adipokine, Albinterferon, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL5, CCL6, CCL7, CCL8, CCL9, Chemokine, Colony-stimulating factor, CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL9, Erythropoietin, Gc-MAF, Granulocyte colony-stimulating factor, Granulocyte macrophage colony-stimulating factor, Hepatocyte growth factor, IL 10 family of cytokines, IL 17 family of cytokines, IL1A, IL1B, Inflammasome, Interferome, Interferon, Interferon beta 1a, Interferon beta 1b, Interferon gamma, Interferon type I, Interferon type II, Interferon type III, Interleukin, Interleukin 1 family, Interleukin 1 receptor antagonist, Interleukin 10, Interleukin 12, Interleukin 12 subunit beta, Interleukin 13, Interleukin 15, Interleukin 16, Interleukin 2, Interleukin 23, Interleukin 23 subunit alpha, Interleukin 34, Interleukin 35, Interleukin 6, Interleukin 7, Interleukin 8, Interleukin 36, Leukemia inhibitory factor, Leukocyte-promoting factor, Lymphokine, Lymphotoxin, Lymphotoxin alpha, Lymphotoxin beta, Macrophage colony-stimulating factor, Macrophage inflammatory protein, Macrophage-activating factor, Monokine, Myokine, Myonectin, Nicotinamide phosphoribosyltransferase, Oncostatin M, Oprelvekin, Platelet factor 4, Proinflammatory cytokine, Promegapoietin, RANKL, Stromal cell-derived factor 1, Talimogene laherparepvec, Tumor necrosis factor alpha, Tumor necrosis factors, XCL1, XCL2, GM-CSF, or XCR1 or any combination thereof. In some embodiments of the methods of making genetically modified T-cells, a transduced population of CD8+ expressing T-cells and/or CD4+ expressing T-cells is contacted with at least one cytokine so as to generate a transduced, cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells. In some embodiments, the at least one cytokine utilized comprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2 or IL-21 or any combination thereof. In some embodiments, the period of contact with the cytokine is at least one day, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that is within a range of times defined by any two of the aforementioned time points.

As used herein, “interleukins” or IL are cytokines that the immune system depends largely upon. Examples of interleukins, which can be utilized herein, for example, include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36 or any combination thereof. Contacting T-cells with interleukins can have effects that promote, support, induce, or improve engraftment fitness of the cells. IL-1, for example can function in the maturation & proliferation of T-cells. IL-2, for example, can stimulate growth and differentiation of T-cell response. IL-3, for example, can promote differentiation and proliferation of myeloid progenitor cells. IL-4, for example, can promote proliferation and differentiation. IL-7, for example, can promote differentiation and proliferation of lymphoid progenitor cells, involved in B, T, and NK cell survival, development, and homeostasis. IL-15, for example, can induce production of natural killer cells. IL-21, for example, co-stimulates activation and proliferation of CD8+ T-cells, augments NK cytotoxicity, augments CD40-driven B cell proliferation, differentiation and isotype switching, and/or promotes differentiation of Th17 cells.

As used herein, “propagating cells” or propagation refers to steps to allow proliferation, expansion, growth and reproduction of cells. For example, cultures of CD8+ T-cells and CD4+ T-cells can typically be incubated under conditions that are suitable for the growth and proliferation of T lymphocytes. In some embodiments of the method of making genetically modified T-cells, which have a chimeric antigen receptor, the CD4+ expressing T-cells are propagated for at least 1 day and may be propagated for 20 days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that is within a range defined by any two of the aforementioned time periods. In some embodiments of the method of making genetically modified T-cells, which have a chimeric antigen receptor, the CD8+ expressing T-cells are propagated for at least 1 day and may be propagated for 20 days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that is within a range defined by any two of the aforementioned time periods.

In another alternative, the expansion method or propagation can further comprise adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least 0.5 ng/ml). In another alternative, the method of making genetically modified T-cells, which have a chimeric antigen receptor method can further comprise adding IL-2, IL-15, or IL-21 or any combination thereof to the culture medium (e.g., wherein the concentration of IL-2 is at least 10 units/mL). In another alternative, the method of making genetically modified T-cells, which have a chimeric antigen receptor method can further comprise adding IL-7, IL-15, or IL21 or any combination thereof to the culture medium (e.g., wherein the concentration of IL-2 is at least 10 units/mL). After isolation of T lymphocytes, both cytotoxic and helper T lymphocytes can be sorted into naïve, memory, and effector T-cell subpopulations either before or after expansion.

As used herein, “genetically modified immune cells” or “Genetically engineered cells” are made by a process called genetic engineering, which can include but is not limited to manipulating a cell's own genome or inserting a new nucleic acid into a cell. In some embodiments, these cells can be macrophages and can also be referred to as genetically engineered macrophages (GEMs). These techniques can be used to change the genetic makeup of the cell, and can include inserting a vector encoding a gene of interest into a cell, and genome editing using RNAi systems, meganucleases, zinc finger nucleases, transcription activator like effector nucleases (TALENS), or CRISPRs. Without being limiting, the vectors encoding the gene of interest can be a viral vector, DNA or an mRNA. In some embodiments, described herein, genetically modified immune cells are provided. In some embodiments, the genetically modified immune cells are made using genome editing proteins or systems, such as for example, meganucleases, zinc finger nucleases, transcription activator like effector nucleases (TALENS), CRISPR/VP64-Cas9 systems or CRISPR/CAS9 systems.

Some embodiments include polypeptide sequences or conservative variations thereof, such as conservative substitutions in a polypeptide sequence. In some embodiments, “conservative amino acid substitution” refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in TABLE 2.

TABLE 2

Family
Amino Acids

non-polar
Trp, Phe, Met, Leu, Ile, Val, Ala, Pro

uncharged polar
Gly, Ser, Thr, Asn, Gln, Tyr, Cys

acidic/negatively charged
Asp, Glu

basic/positively charged
Arg, Lys, His

Beta-branched
Thr, Val, Ile

residues that influence chain
Gly, Pro

orientation

aromatic
Trp, Tyr, Phe, His

Certain Nucleic Acids

Some embodiments the methods and compositions provided herein include one or more nucleic acids encoding a CAR capable of specifically binding to a FGFR4 protein, such as an extracellular domain of a FGFR4 protein, such as an IgIII membrane-proximal domain of a FGFR4 protein. In some embodiments, the CAR comprises a ligand binding domain capable of specifically binding to a FGFR4 protein; a transmembrane domain; and an intracellular signalling domain.

In some embodiments, the ligand binding domain comprises a complementarity-determining region (CDR) derived from a binding moiety polypeptide, such as a binding moiety polypeptide capable of specifically binding to a FGFR4 protein, such as an extracellular domain of a FGFR4 protein, such as an IgIII membrane-proximal domain of a FGFR4 protein. In some embodiments, the ligand binding domain comprises an antigen-binding fragment of an antibody, an scFv, or a variable heavy chain (VH) domain and a variable heavy chain (VL) domain. In some embodiments, the VH domain and the VL domain are linked via a linker, such as a triple repeat of the amino acid sequence “GGGGS”. In some embodiments, the ligand binding domain comprises a VH-VL domains in which the VH domain precedes the VL domain in the polypeptide. In some embodiments, the ligand binding domain comprises a VL-VH domains in which the VL domain precedes the VH domain in the polypeptide.

In some embodiments, the ligand binding domain comprises, consists essentially of, or consists of a VH domain. In some embodiments, the ligand binding domain lacks a VL domain. In some embodiments, the ligand binding domain can consist essentially of a VH domain and no more than 10 other amino acids. In some embodiments, the ligand binding domain can consist essentially of a VH domain and no more than 5 other amino acids. In some embodiments, the ligand binding domain can consist essentially of a VH domain and no more than 3 other amino acids.

In some embodiments, the ligand binding domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:01-22. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:01-04, 06, 07, 11-14, 17, 18 and 20. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:06. In some embodiments, the ligand binding domain comprises the amino acid sequence of SEQ ID NO:06.

In some embodiments, the ligand binding domain comprises a CDR of a binding moiety polypeptide, such as a binding moiety polypeptide which specifically binds to a FGFR4 protein, such as an extracellular domain of a FGFR4 protein, such as an IgM membrane-proximal domain of a FGFR4 protein. In some embodiments, the ligand binding domain can include a heavy chain CDR. In some embodiments, the ligand binding domain can include two heavy chain CDRs. In some embodiments, the ligand binding domain can include three heavy chain CDRs. In some embodiments, the ligand binding domain can include a light chain CDR. In some embodiments, the ligand binding domain can include two light chain CDRs. In some embodiments, the ligand binding domain can include three light chain CDRs. In some embodiments, the ligand binding domain can include a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and/or a light chain CDR3, derived from a binding moiety polypeptide which specifically binds to a FGFR4 protein, such as an extracellular domain of a FGFR4 protein, such as an IgIII membrane-proximal domain of a FGFR4 protein. In some embodiments, the ligand binding domain comprises a CDR domain comprising or consisting of a CDR sequence comprising an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:42-149. In some embodiments, the ligand binding domain comprises a CDR domain comprising or consisting of a CDR sequence comprising an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:41-53, 57-62, 78-101, 114-125 and 132-137. In some embodiments, the ligand binding domain comprises a CDR domain comprising or consisting of a CDR sequence comprising an amino acid sequence having 0-4 conservative amino acid substitutions of an amino acid sequence of any one of SEQ ID NOs:57-59.

In some embodiments, the transmembrane domain comprises a domain selected from a CD8 transmembrane domain and a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

In some embodiments, the intracellular signalling domain comprises a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and CD3-zeta. In some embodiments, the intracellular signalling domain comprises a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, and B7-H3 in combination with a CD3-zeta domain. In some embodiments, the intracellular signalling domain comprises a 4-1BB costimulatory domain in combination with a CD3-zeta domain. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, the CD3-zeta domain comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31.

In some embodiments, the CAR comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a CAR listed in TABLE 5 or TABLE 6. In some embodiments, the CAR comprises an amino acid sequence having at least or equal to 90%, 91%, 92%, 93%, 94% 95%, 96% 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:41.

In some embodiments, the CAR also includes a spacer located between the ligand binding domain and the transmembrane domain. In some embodiments, the spacer comprises a short (S), medium (M), or long (L) spacer. Examples of spacers are listed in TABLE 5.

In some embodiments, the nucleic acid also includes a promoter operably linked to the polynucleotide encoding a chimeric antigen receptor. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the constitutive promoter comprises an EF1α promoter. In some embodiments, the promoter comprises an inducible promoter.

In some embodiments, the nucleic acid also includes a polynucleotide encoding a cell-surface selectable marker. In some embodiments, the cell-surface selectable marker is selected from a truncated EGFR polypeptide (EGFRt) or a truncated Her2 polypeptide (Her2t). In some embodiments, the nucleic acid also includes a ribosome skip sequence located between a polynucleotide encoding the intracellular signalling domain and the polynucleotide encoding a cell-surface selectable marker. In some embodiments, the ribosome skip sequence is selected from the group consisting of a P2A sequence, a T2A sequence, an E2A sequence, and an F2A sequence.

In some embodiments, the nucleic acid also includes a polynucleotide encoding a suicide gene system. In some embodiments, the suicide gene system is selected from a herpes simplex virus thymidine kinase/ganciclovir (HSVTK/GCV) suicide gene system, or an inducible caspase suicide gene system.

Some embodiments of the methods and compositions provided herein include a vector comprising any one of the nucleic acids provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector, foamy viral vector, retroviral vector, an adenoviral vector, or an adenovirus associated viral vector. In some embodiments, the vector is a transposon, integrase vector system, or an mRNA vector.

Certain sequences useful with embodiments provided herein are listed in TABLE 3, TABLE 4, TABLE 5 or TABLE 6. In TABLE 3, CDR sequences within the listed binding polypeptide sequences are underlined. Heavy chain CDRs and light chain CDRs can be readily determined from TABLE 3. For example, sequences of screen ‘VH domain only’ include heavy chain CDR1, CDR2, and CDR3; a binder ‘VLVH’ includes a first three light chain CDRs, and a second three heavy chain CDRs; and a binder ‘VHVL’ includes a first three heavy chain CDRs, and a second three light chain CDRs. TABLE 4 lists CDR sequences from TABLE 3.

TABLE 3

FGFR4

binding

poly-

peptide

SEQ ID

(binder)
Screen
NO:
Sequence

E9
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCTVSGFTFSSYA

domain
NO: 01

LGWVRQAPGKGLEWIGYIYHKGDTNYNPSLKS

only

LVTISRDNSKNTLYLQMNSLRAEDTALYYCAK

VRWVSSTSTLGFDYWGQGTLVTVSS

A4
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCVASGFTFISYA

domain
NO: 02

MSWVRQAPGKGLEWMAIIYPDDAGTMYSPSF

only

QGQVTISRDNSKNTLYLQMNSLRAEDTALYYC

ARVTMVRGVIGFDPWGQGTLVTVSS

E61
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCTFSGFTFSSYA

domain
NO: 03

MSWVRQAPGKGLEWMGIIYPGDSNTIYSPSFQ

only

GQVTISRDNSKNTLYLQMNSLRAEDTATYYCA

RSGAYDGRIWFDPWGQGTLVTVSS

A7
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNY

domain
NO: 04

GMSWVRQAPGKGLEWIGYIYHTGNTYYNPSL

only

KSRVTISRDNSKNTLYLQMNSLRAEDTAIYYC

AREGVMGGYHTDPNYYYYGMDVWGQGTTVT

VSS

D92
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYG

domain
NO: 05

MHWVRQAPGKGLEWIGYSYYSGSTYYNPSLK

only

SLVTISRDNSKNTLYLQMNSLRAEDTATYYCA

RDGSSWYSFPYYYGMDVWGQGTTVTVSS

D8
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNY

domain
NO: 06

GMSWVRQAPGKALEWIGEINHSGSTNYNPSLK

only

SRVTISRDNSKNTLYLQMNSLRAEDTAIYYCRG

GWVGYWGQGTLVTVSS

F11
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNY

domain
NO: 07

GMSWVRQAPGQGLEWVSYISNGGGTMYYAD

only

SVKGRFTISRDNSKNTLYLQMNSLRAEDTATY

YCTRAPYCSGGDCYPMSTHELDSWGQGTLVT

VSS

C52
VH
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGFTLSSY

domain
NO: 08

WMSWVRQAPGKGLEWIGVIYYSGATYYNPSL

only

KSLVTISRDNSKNTLYLQMNSLRAEDTAVYYC

ARGALDYFDLWGQGALVTVSS

H4-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 09
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDVGVYYCLQITHWPHTFGQ

GTKLEIKRGGGGSGGGGSGGGGSQVQLVESGG

GLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP

GKGLEWVSYISSSGSTIYYADSVKGRFTISRDN

AKNSLYLQMNSLRAEDTAMYYCARDAPDLLA

VPTVIFSGYHYGADVWGQGATVTVSS

H4-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 10

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAMYYC

ARDAPDLLAVPTVIFSGYHYGADVWGQGATV

TVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKL

LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DVGVYYCLQITHWPHTFGQGTKLEIKR

G6-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 11
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDAATYYCQHYGTFGQGTKL

EIKRGGGGSGGGGSGGGGSQVQLVESGGGLV

KPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG

LEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARHKTSLNDYDF

WSGYLLDYWGQGTLVTVSS

G6-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 12

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

ARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD

RVTITCRASQSISSYLNWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAA

TYYCQHYGTFGQGTKLEIKR

C9-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 13
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQRSNWPPTFGQ

GTKLEIKRGGGGSGGGGSGGGGSQVQLVESGG

GLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP

GKGLEWVSYISSSGSTIYYADSVKGRFTISRDN

AKNSLYLQMNSLRAEDTAKYYCATAVFSLGPE

GYYYYMDVWGKGTTVTVSS

C9-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVLH

NO: 14

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAKYYC

ATAVFSLGPEGYYYYMDVWGKGTTVTVSSGG

GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRV

TITCRASQSISSYLNWYQQKPGKAPKLLIYAAS

SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVY

YCQQRSNWPPTFGQGTKLEIKR

D7-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 15
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDIAAYYCQHYGTFGQGTKL

EIKRGGGGSGGGGSGGGGSQVQLVESGGGLV

KPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG

LEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARDGYPLGGGYD

FWSGYYPDYWGQGTLVTVSS

D7-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 16

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

ARDGYPLGGGYDFWSGYYPDYWGQGTLVTVS

SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG

DRVTITCRASQSISSYLNWYQQKPGKAPKLLIY

AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIA

AYYCQHYGTFGQGTKLEIKR

B7-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 17
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDFAVYYCQHYGTFGQGTKV

EIKRGGGGSGGGGSGGGGSQVQLVESGGGLV

KPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG

LEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAMYYCARHKTSLNDYDF

WSGYLLDYWGQGTLVTVSS

B7-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 18
YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAMYYC

ARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD

RVTITCRASQSISSYLNWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA

VYYCQHYGTFGQGTKVEIKR

A6-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 19
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDAATYYCQRYGTFGQGTKV

DIKRGGGGSGGGGSGGGGSQVQLVESGGGLV

KPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG

LEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARHKTSLNDYDF

WSGYLLDYWGQGTLVTVSS

A6-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 20

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

ARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD

RVTITCRASQSISSYLNWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAA

TYYCQRYGTFGQGTKVDIKR

C1-
F(ab)
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN

VLVH
screen
NO: 21
WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDAATYYCQHYGTFGQGTK

VEIKRGGGGSGGGGSGGGGSQVQLVESGGGL

VKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

GLEWVSYISSSGSTIYYADSVKGRFTISRDNAK

NSLYLQMNSLRAEDTAVYYCARHAPHKHYDF

WSGYYPDYWGQGTLVTVSS

C1-
—
SEQ ID
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDY

VHVL

NO: 22

YMSWIRQAPGKGLEWVSYISSSGSTIYYADSV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

ARHAPHKHYDFWSGYYPDYWGQGTLVTVSSG

GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR

VTITCRASQSISSYLNWYQQKPGKAPKLLIYAA

SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAAT

YYCQHYGTFGQGTKVEIKR

TABLE 4

FGFR4

binding

poly-

peptide

(binder)
SEQ ID NO
CDR Sequences

E9
SEQ ID NO: 42
SYALG

SEQ ID NO: 43
YIYHKGDTNYNPSLKS

SEQ ID NO: 44
VRWVSSTSTLGFDY

A4
SEQ ID NO: 45
SYAMS

SEQ ID NO: 46
IIYPDDAGTMYSPSFQG

SEQ ID NO: 47
VTMVRGVIGFDP

E61
SEQ ID NO: 48
SYAMS

SEQ ID NO: 49
MGIIYPGDSNTIYSPSFQG

SEQ ID NO: 50
SGAYDGRIWFDP

A7
SEQ ID NO: 51
NYGMS

SEQ ID NO: 52
YIYHTGNTYYNPSLKS

SEQ ID NO: 53
EGVMGGYHTDPNYYYYGMDV

D92
SEQ ID NO: 54
SYGMH

SEQ ID NO: 55
YSYYSGSTYYNPSLKS

SEQ ID NO: 56
DGSSWYSFPYYYGMDV

D8
SEQ ID NO: 57
NYGMS

SEQ ID NO: 58
EINHSGSTNYNPSLKS

SEQ ID NO: 59
RGGWVGY

F11
SEQ ID NO: 60
NYGMS

SEQ ID NO: 61
YISNGGGTMYYADSVKG

SEQ ID NO: 62
TRAPYCSGGDCYPMSTHELDS

C52
SEQ ID NO: 63
SYWMS

SEQ ID NO: 64
VIYYSGATYYNPSLKS

SEQ ID NO: 65
GALDYFDL

H4-VLVH
SEQ ID NO: 66
RASQSISSYLN

SEQ ID NO: 67
AASSLQS

SEQ ID NO: 68
LQITHWPHT

SEQ ID NO: 69
DYYMS

SEQ ID NO: 70
YISSSGSTIYYADSVKG

SEQ ID NO: 71
DAPDLLAVPTVIFSGYHYGADV

H4-VHVL
SEQ ID NO: 72
DYYMS

SEQ ID NO: 73
YISSSGSTIYYADSVKG

SEQ ID NO: 74
DAPDLLAVPTVIFSGYHYGADV

SEQ ID NO: 75
RASQSISSYLN

SEQ ID NO: 76
AASSLQS

SEQ ID NO: 77
LQITHWPHT

G6-VLVH
SEQ ID NO: 78
RASQSISSYLN

SEQ ID NO: 79
AASSLQS

SEQ ID NO: 80
QHYGT

SEQ ID NO: 81
DYYMS

SEQ ID NO: 82
YISSSGSTIYYADSVKG

SEQ ID NO: 83
HKTSLNDYDFWSGYLLDY

G6-VHVL
SEQ ID NO: 84
DYYMS

SEQ ID NO: 85
YISSSGSTIYYADSVKG

SEQ ID NO: 86
HKTSLNDYDFWSGYLLDY

SEQ ID NO: 87
RASQSISSYLN

SEQ ID NO: 88
AASSLQS

SEQ ID NO: 89
QHYGT

C9-VLVH
SEQ ID NO: 90
RASQSISSYLN

SEQ ID NO: 91
AASSLQS

SEQ ID NO: 92
QQRSNWPPT

SEQ ID NO: 93
DYYMS

SEQ ID NO: 94
YISSSGSTIYYADSVKG

SEQ ID NO: 95
AVFSLGPEGYYYYMDV

C9-VHVLH
SEQ ID NO: 96
DYYMS

SEQ ID NO: 97
YISSSGSTIYYADSVKG

SEQ ID NO: 98
AVFSLGPEGYYYYMDV

SEQ ID NO: 99
RASQSISSYLN

SEQ ID NO: 100
AASSLQS

SEQ ID NO: 101
QQRSNWPPT

D7-VLVH
SEQ ID NO: 102
RASQSISSYLN

SEQ ID NO: 103
AASSLQS

SEQ ID NO: 104
QHYGT

SEQ ID NO: 105
DYYMS

SEQ ID NO: 106
YISSSGSTIYYADSVKG

SEQ ID NO: 107
DGYPLGGGYDFWSGYYPDY

D7-VHVL
SEQ ID NO: 108
DYYMS

SEQ ID NO: 109
YISSSGSTIYYADSVKG

SEQ ID NO: 110
DGYPLGGGYDFWSGYYPDY

SEQ ID NO: 111
RASQSISSYLN

SEQ ID NO: 112
AASSLQS

SEQ ID NO: 113
QHYGT

B7-VLVH
SEQ ID NO: 114
RASQSISSYLN

SEQ ID NO: 115
AASSLQS

SEQ ID NO: 116
QHYGT

SEQ ID NO: 117
DYYMS

SEQ ID NO: 118
YISSSGSTIYYADSVKG

SEQ ID NO: 119
HKTSLNDYDFWSGYLLDY

B7-VHVL
SEQ ID NO: 120
DYYMS

SEQ ID NO: 121
YISSSGSTIYYADSVKG

SEQ ID NO: 122
HKTSLNDYDFWSGYLLDY

SEQ ID NO: 123
RASQSISSYLN

SEQ ID NO: 124
AASSLQS

SEQ ID NO: 125
QHYGT

A6-VLVH
SEQ ID NO: 126
RASQSISSYLN

SEQ ID NO: 127
AASSLQS

SEQ ID NO: 128
QRYGT

SEQ ID NO: 129
DYYMS

SEQ ID NO: 130
YISSSGSTIYYADSVKG

SEQ ID NO: 131
HKTSLNDYDFWSGYLLDY

A6-VHVL
SEQ ID NO: 132
DYYMS

SEQ ID NO: 133
YISSSGSTIYYADSVKG

SEQ ID NO: 134
HKTSLNDYDFWSGYLLDY

SEQ ID NO: 135
RASQSISSYLN

SEQ ID NO: 136
AASSLQS

SEQ ID NO: 137
QRYGT

C1-VLVH
SEQ ID NO: 138
RASQSISSYLN

SEQ ID NO: 139
AASSLQS

SEQ ID NO: 140
QHYGT

SEQ ID NO: 141
DYYMS

SEQ ID NO: 142
YISSSGSTIYYADSVKG

SEQ ID NO: 143
HAPHKHYDFWSGYYPDY

C1-VHVL
SEQ ID NO: 144
DYYMS

SEQ ID NO: 145
YISSSGSTIYYADSVKG

SEQ ID NO: 146
HAPHKHYDFWSGYYPDY

SEQ ID NO: 147
RASQSISSYLN

SEQ ID NO: 148
AASSLQS

SEQ ID NO: 149
QHYGT

TABLE 5

Feature

SEQ ID NO
Sequence

Leader peptide
MLLLVTSLLLCELPHPAFLLIP

SEQ ID NO: 23

CD8 linker and
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

transmembrane
DIYIWAPLAGTCGVLLLSLVITLYC

domains

SEQ ID NO: 24

CD8 linker and
ACCACTACTCCGGCACCTAGGCCCCCCACTCCGGCACCGACC

transmembrane
ATTGCATCACAACCACTGAGTCTTAGACCTGAAGCCTGTCGA

domains
CCCGCAGCTGGGGGGGCAGTCCACACACGGGGATTGGATTT

SEQ ID NO: 25
CGCCTGCGATATATACATTTGGGCGCCACTGGCAGGCACCTG

CGGGGTCCTGCTCTTGTCCCTTGTCATCACCCTGTACTGT

CD28tm
MFWVLVVVGGVLACYSLLVTVAFIIFWV

SEQ ID NO: 26

CD28tm
ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGC

SEQ ID NO: 27
TACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG

CD28
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

cytoplasmic

SEQ ID NO: 28

4-1BB signaling
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

domain

SEQ ID NO: 29

4-1BB signaling
AAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCAAACAGCC

domain
ATTCATGAGGCCGGTCCAGACCACTCAGGAGGAGGATGGAT

SEQ ID NO: 30
GCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGGGTGTGAA

CTC

CD3-zeta chain
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD

signaling
PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK

domain
GHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 31

CD3-zeta chain
CGAGTTAAATTTTCTCGCAGTGCTGATGCCCCAGCGTATAAA

signaling
CAGGGTCAGAACCAACTGTATAATGAACTCAATTTGGGGAG

domain
GCGAGAGGAGTATGATGTATTGGACAAGCGGCGGGGGCGGG

SEQ ID NO: 32
ATCCTGAAATGGGCGGTAAGCCAAGGCGCAAGAATCCACAG

GAGGGTTTGTATAATGAGTTGCAGAAGGATAAAATGGCGGA

AGCGTATTCAGAAATCGGAATGAAGGGAGAGCGCCGCAGAG

GGAAAGGGCATGATGGCCTCTACCAAGGTTTGTCAACAGCG

ACCAAGGATACCTATGACGCACTTCATATGCAAGCACTGCC

CCCCAGG

T2A
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATG

SEQ ID NO: 33
CGGTGACGTGGAGGAGAATCCCGGCCCTAGG

EGFRt
CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGA

SEQ ID NO: 34
CTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAA

CTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGC

ATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCC

ACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAG

GGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACC

TCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACC

AAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAAC

ATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGAT

GGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGC

AAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTC

AGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGC

AAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGA

GGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCC

GGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAAC

CTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGA

GTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGA

ACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAG

TGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGC

CCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAA

GTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAA

ACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGT

CCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGAT

GGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGAT

CGGCCTCTTCATG

S (small) spacer
ESKYGPPCPPCP

SEQ ID NO: 35

S (small) spacer
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT

SEQ ID NO: 36

M (medium)
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF

spacer
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR

SEQ ID NO: 37
WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

M (medium)
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTGGCCAG

spacer
CCTAGAGAACCCCAGGTGTACACCCTGCCTCCCAGCCAGGA

SEQ ID NO: 38
AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCA

AAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGC

AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT

GCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGAC

CGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTCAGCT

GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG

AAGTCCCTGAGCCTGAGCCTGGGCAAG

L (long) spacer
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

SEQ ID NO: 39
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK

L (long) spacer
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTGCCCCC

SEQ ID NO: 40
GAGTTCGACGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAG

CCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGAC

CTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCCGAGGTCC

AGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCC

AAGACCAAGCCCAGAGAGGAACAGTTCCAGAGCACCTACCG

GGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAA

CGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGC

CCAGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCCA

GCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGA

AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGA

AGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC

AACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGT

GCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGA

CCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGC

TGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCA

GAAGAGCCTGAGCCTGTCCCTGGGCAAG

RJ146 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(D8/CD8 TM/
AASGFTFSNYGMSWVRQAPGKALEWIGEINHSGSTNYNPSLKS

4-1BB/CD3zeta)
RVTISRDNSKNTLYLQMNSLRAEDTAIYYCRGGWVGYWGQGT

SEQ ID NO: 41
LVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH

TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA

YKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST

ATKDTYDALHMQALPPR

Certain CARs Specific for FGFR4

Some embodiments of the methods and compositions provided herein include a CAR specific for FGFR4. In some such embodiments, the CAR is encoded by a nucleic acid provided herein. The CAR can be encoded by the nucleic acid of any one of the embodiments herein or the vector of any one of the embodiments herein.

Certain Cells for CAR T Cell Expression and Therapy

Some embodiments of the methods and compositions provided herein include a cell comprising a CAR specific for FGFR4, as described in any one of the embodiments herein, or a nucleic acid encoding a CAR specific for FGFR4, as described in any one of the embodiments herein. In some embodiments, the cell is from a donor that is the same as the recipient of the therapy (e.g., autologous CAR T cell therapy), or related or unrelated to the subject in need of CAR T cell therapy. In some embodiments, the cell is from the subject in need of CAR T cell therapy. In some embodiments, the cell is allogeneic to a subject in need of CAR T cell therapy. In some embodiments, the cell is human.

In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4+ T-cell or a CD8+ T-cell. In some embodiments, the cell is a CD8+ cytotoxic T-cell selected from the group consisting of a naïve CD8+ T-cell, a CD8+ memory T-cell, a central memory CD8+ T-cell, a regulatory CD8+ T-cell, an IPS derived CD8+ T-cell, an effector memory CD8+ T-cell, and a bulk CD8+ T-cell. In some embodiments, the cell is a CD4+ T helper cell selected from the group consisting of a naïve CD4+ T-cell, a CD4+ memory T-cell, a central memory CD4+ T-cell, a regulatory CD4+ T-cell, an IPS derived CD4+ T-cell, an effector memory CD4+ T-cell, and a bulk CD4+ T-cell. In some embodiments, the cell is a precursor T-cell, or a hematopoietic stem cell.

Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising a cell comprising a CAR specific for FGFR4 as described in any one of the embodiments herein, or a nucleic acid encoding a CAR specific for FGFR4 as described in any one of the embodiments herein, and a pharmaceutically acceptable excipient.

Certain Methods of Preparing Donor Cells

Some embodiments of the methods and compositions provided herein include methods of preparing a population of cells comprising a CAR specific for FGFR4, such as a population of cells for an infusion. Some embodiments include obtaining a cell from a subject, or a cell from another matched to be compatible to the subject for cellular therapy. Some embodiments also include introducing any one of the vectors providing herein containing a CAR specific for FGFR4 into the cell. Some embodiments also include expanding the cell and isolating the cell. Some embodiments include culturing the cell in the presence of an agent selected from an anti-CD3, an anti-CD28, or a cytokine, such as IL-2.

Certain Methods of Therapy

Some embodiments of the methods and compositions provided herein include methods of therapy, such as methods of treating, inhibiting or ameliorating a cancer in a subject. In some embodiments, the cancer comprises a cancer cell expressing FGFR4. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a cell expressing a protein selected from a PAX3-FOXO1 fusion gene product, and a PAX7-FOXO1 fusion gene product. In some embodiments, the cancer comprises a cell comprising a mutation in a gene selected from TP53, RAS, PI3K3CA, CTNNB1, or FGFR4. In some embodiments, the cancer comprises a rhabdomyosarcoma. In some embodiments, the subject is human.

Certain sequences useful with embodiments provided herein are listed in TABLE 6.

TABLE 6

Feature

SEQ ID NO
Sequence

E9 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 150
TGGCGGATCTCTGAGACTGAGCTGTACCGTGTCCGGCTTCAC

CTTCAGCTCTTATGCCCTCGGCTGGGTCCGACAGGCTCCTGG

AAAAGGACTGGAATGGATCGGCTACATCTACCACAAGGGCG

ACACCAACTACAACCCCAGCCTGAAGTCCCTGGTCACCATC

AGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAA

CAGCCTGAGAGCCGAGGACACAGCCCTGTACTACTGCGCCA

AAGTCCGCTGGGTGTCCAGCACAAGCACCCTGGGCTTTGATT

ATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT

RJ141 CAR:
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCT

(E9/CD8 TM/
VSGFTFSSYALGWVRQAPGKGLEWIGYIYHKGDTNYNPSLKSL

4-1BB/CD3zeta)
VTISRDNSKNTLYLQMNSLRAEDTALYYCAKVRWVSSTSTLGF

SEQ ID NO: 151
DYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACR

PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR

GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF

SRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

GLYQGLSTATKDTYDALHMQALPPR

RJ141 CAR:
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(B9/CD8 TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 152
AGCTGTACCGTGTCCGGCTTCACCTTCAGCTCTTATGCCCTC

GGCTGGGTCCGACAGGCTCCTGGAAAAGGACTGGAATGGAT

CGGCTACATCTACCACAAGGGCGACACCAACTACAACCCCA

GCCTGAAGTCCCTGGTCACCATCAGCAGAGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGA

CACAGCCCTGTACTACTGCGCCAAAGTCCGCTGGGTGTCCAG

CACAAGCACCCTGGGCTTTGATTATTGGGGCCAGGGCACAC

TGGTCACCGTGTCATCTGCGGCCGCAACCACTACTCCGGCAC

CTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACCAC

TGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGG

GCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATATAC

ATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTG

TCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAA

GTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCA

GACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGG

AAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCT

CGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCA

ACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATG

ATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGGGC

GGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAA

TGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAA

TCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGA

TGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCT

ATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

A4 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 153
TGGCGGATCTCTGAGACTGAGCTGTGTGGCCAGCGGCTTCAC

CTTCATCAGCTACGCCATGAGCTGGGTCCGACAGGCCCCTGG

AAAAGGCCTTGAGTGGATGGCCATTATCTACCCCGATGACG

CCGGCACCATGTACAGCCCTAGCTTTCAGGGCCAAGTGACC

ATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGAT

GAACAGCCTGAGAGCCGAGGACACAGCCCTGTACTACTGTG

CCAGAGTGACAATGGTCCGAGGCGTGATCGGCTTCGATCCTT

GGGGACAGGGAACCCTGGTCACCGTTTCTTCT

RJ142 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(A4/CD8 TM/
VASGFTFISYAMSWVRQAPGKGLEWMAIIYPDDAGTMYSPSFQ

4-1BB/CD3zeta)
GQVTISRDNSKNTLYLQMNSLRAEDTALYYCARVTMVRGVIG

SEQ ID NO: 154
FDPWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACR

PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR

GRKKLLYIFKQPFMRPVQTTQEEDGCCRFPEEEEGGCELRVKF

SRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

GLYQGLSTATKDTYDALHMQALPPR

RJ142 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(A4/CD8 TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 155
AGCTGTGTGGCCAGCGGCTTCACCTTCATCAGCTACGCCATG

AGCTGGGTCCGACAGGCCCCTGGAAAAGGCCTTGAGTGGAT

GGCCATTATCTACCCCGATGACGCCGGCACCATGTACAGCCC

TAGCTTTCAGGGCCAAGTGACCATCAGCCGGGACAACAGCA

AGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAG

GACACAGCCCTGTACTACTGTGCCAGAGTGACAATGGTCCG

AGGCGTGATCGGCTTCGATCCTTGGGGACAGGGAACCCTGG

TCACCGTTTCTTCTGCGGCCGCAACCACTACTCCGGCACCTA

GGCCCCCCACTCCGGCACCGACCATTGCATCACAACCACTG

AGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGGGC

AGTCCACACACGGGGATTGGATTTCGCCTGCGATATATACAT

TTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTGTC

CCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAAGT

TGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCAGA

CCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGGAA

GAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCTCG

CAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCAAC

TGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATGAT

GTATTGGACAAGCGGCGGGGGGGGGATCCTGAAATGGGCGG

TAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAATG

AGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAATC

GGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGATG

GCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCTAT

GACGCACTTCATATGCAAGCACTGCCCCCCAGG

B61 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 156
TGGCGGATCTCTGAGACTGTCCTGCACCTTCAGCGGCTTCAC

CTTCTCCAGCTACGCCATGAGCTGGGTCCGACAGGCTCCTGG

CAAAGGCCTTGAGTGGATGGGCATCATCTACCCCGGCGACA

GCAACACCATCTACAGCCCTAGCTTCCAGGGCCAAGTGACC

ATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGAT

GAACAGCCTGAGAGCCGAGGACACCGCCACCTACTACTGTG

CTAGAAGCGGCGCCTACGACGGCCGGATTTGGTTTGATCCTT

GGGGCCAGGGCACCCTGGTCACAGTTTCTTCT

RJ143 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCT

(B61/CD8TM/
FSGFTFSSYAMSWVRQAPGKGLEWMGIIYPGDSNTIYSPSFQGQ

4-1BB/CD3zeta)
VTISRDNSKNTLYLQMNSLRAEDTATYYCARSGAYDGRIWFDP

SEQ ID NO: 157
WGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPA

AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR

KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR

SADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG

KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL

YQGLSTATKDTYDALHMQALPPR

RJ143 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(E61/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 158
TCCTGCACCTTCAGCGGCTTCACCTTCTCCAGCTACGCCATG

AGCTGGGTCCGACAGGCTCCTGGCAAAGGCCTTGAGTGGAT

GGGCATCATCTACCCCGGCGACAGCAACACCATCTACAGCC

CTAGCTTCCAGGGCCAAGTGACCATCAGCCGGGACAACAGC

AAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGA

GGACACCGCCACCTACTACTGTGCTAGAAGCGGCGCCTACG

ACGGCCGGATTTGGTTTGATCCTTGGGGCCAGGGCACCCTGG

TCACAGTTTCTTCTGCGGCCGCAACCACTACTCCGGCACCTA

GGCCCCCCACTCCGGCACCGACCATTGCATCACAACCACTG

AGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGGGC

AGTCCACACACGGGGATTGGATTTCGCCTGCGATATATACAT

TTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTGTC

CCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAAGT

TGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCAGA

CCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGGAA

GAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCTCG

CAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCAAC

TGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATGAT

GTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGGGCGG

TAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAATG

AGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAATC

GGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGATG

GCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCTAT

GACGCACTTCATATGCAAGCACTGCCCCCCAGG

A7 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 159
TGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCAC

CTTCAGCAACTACGGCATGAGCTGGGTCCGACAGGCCCCTG

GAAAAGGCCTTGAGTGGATCGGCTACATCTACCACACCGGC

AACACCTACTACAACCCCAGCCTGAAGTCCAGAGTGACCAT

CAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGA

ACAGCCTGAGAGCCGAGGACACCGCCATCTACTACTGTGCC

AGAGAAGGCGTGATGGGCGGCTACCACACAGACCCCAATTA

CTACTACTATGGCATGGACGTGTGGGGCCAGGGCACCACAG

TGACAGTTTCTTCT

RJ144 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(A7/CD8 TM/
AASGFTFSNYGMSWVRQAPGKGLEWIGYIYHTGNTYYNPSLK

4-1BB/CD3zeta)
SRVTISRDNSKNTLYLQMNSLRAEDTAIYYCAREGVMGGYHT

SEQ ID NO: 160
DPNYYYYGMDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQ

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS

LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDK

RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

RJ144 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(A7/CD8 TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 161
TCTTGTGCCGCCAGCGGCTTCACCTTCAGCAACTACGGCATG

AGCTGGGTCCGACAGGCCCCTGGAAAAGGCCTTGAGTGGAT

CGGCTACATCTACCACACCGGCAACACCTACTACAACCCCA

GCCTGAAGTCCAGAGTGACCATCAGCCGGGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGA

CACCGCCATCTACTACTGTGCCAGAGAAGGCGTGATGGGCG

GCTACCACACAGACCCCAATTACTACTACTATGGCATGGAC

GTGTGGGGCCAGGGCACCACAGTGACAGTTTCTTCTGCGGC

CGCAACCACTACTCCGGCACCTAGGCCCCCCACTCCGGCACC

GACCATTGCATCACAACCACTGAGTCTTAGACCTGAAGCCTG

TCGACCCGCAGCTGGGGGGGCAGTCCACACACGGGGATTGG

ATTTCGCCTGCGATATATACATTTGGGCGCCACTGGCAGGCA

CCTGCGGGGTCCTGCTCTTGTCCCTTGTCATCACCCTGTACTG

TAAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCAAACAGC

CATTCATGAGGCCGGTCCAGACCACTCAGGAGGAGGATGGA

TGCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGGGTGTGA

ACTCCGAGTTAAATTTTCTCGCAGTGCTGATGCCCCAGCGTA

TAAACAGGGTCAGAACCAACTGTATAATGAACTCAATTTGG

GGAGGCGAGAGGAGTATGATGTATTGGACAAGCGGCGGGG

GCGGGATCCTGAAATGGGCGGTAAGCCAAGGCGCAAGAATC

CACAGGAGGGTTTGTATAATGAGTTGCAGAAGGATAAAATG

GCGGAAGCGTATTCAGAAATCGGAATGAAGGGAGAGCGCCG

CAGAGGGAAAGGGCATGATGGCCTCTACCAAGGTTTGTCAA

CAGCGACCAAGGATACCTATGACGCACTTCATATGCAAGCA

CTGCCCCCCAGG

D92 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 162
TGGCGGATCTCTGAGACTGAGCTGTGTGGCCAGCGGCTTCAC

CTTTAGCAGCTACGGCATGCACTGGGTCCGACAGGCACCTG

GCAAAGGCCTGGAATGGATCGGCTACAGCTACTACAGCGGC

AGCACCTACTACAACCCCAGCCTGAAGTCCCTGGTCACCATC

AGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAA

CAGCCTGAGAGCCGAGGACACCGCCACCTACTATTGTGCCA

GAGATGGCAGCAGCTGGTACAGCTTCCCCTACTACTATGGC

ATGGACGTGTGGGGCCAGGGCACCACAGTGACAGTTTCTTCT

RJ145 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(D92/CD8TM/
VASGFTFSSYGMHWVRQAPGKGLEWIGYSYYSGSTYYNPSLK

4-1BB/CD3zeta)
SLVTISRDNSKNTLYLQMNSLRAEDTATYYCARDGSSWYSFPY

SEQ ID NO: 163
YYGMDVWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRP

EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY

CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR

VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK

GHDGLYQGLSTATKDTYDALHMQALPPR

RJ145 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(D92/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 164
AGCTGTGTGGCCAGCGGCTTCACCTTTAGCAGCTACGGCATG

CACTGGGTCCGACAGGCACCTGGCAAAGGCCTGGAATGGAT

CGGCTACAGCTACTACAGCGGCAGCACCTACTACAACCCCA

GCCTGAAGTCCCTGGTCACCATCAGCAGAGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGA

CACCGCCACCTACTATTGTGCCAGAGATGGCAGCAGCTGGT

ACAGCTTCCCCTACTACTATGGCATGGACGTGTGGGGCCAG

GGCACCACAGTGACAGTTTCTTCTGCGGCCGCAACCACTACT

CCGGCACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCA

CAACCACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCT

GGGGGGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGA

TATATACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCT

GCTCTTGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAG

AAAGAAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCC

GGTCCAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCT

TTCCGGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAA

TTTTCTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAG

AACCAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGA

GTATGATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAA

TGGGCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTG

TATAATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTC

AGAAATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGG

CATGATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGA

TACCTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

D8 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 165
TGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCAC

CTTCAGCAACTACGGCATGAGCTGGGTCCGACAGGCCCCTG

GAAAAGCCCTGGAATGGATCGGCGAGATCAACCACAGCGGC

AGCACCAACTACAACCCCAGCCTGAAGTCCAGAGTGACCAT

CAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGA

ACAGCCTGAGAGCCGAGGACACCGCCATCTACTACTGTAGA

GGCGGCTGGGTCGGATATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGT

RJ146 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(D8/CD8 TM/
AASGFTFSNYGMSWVRQAPGKALEWIGEINHSGSTNYNPSLKS

4-1BB/CD3zeta)
RVTISRDNSKNTLYLQMNSLRAEDTAIYYCRGGWVGYWGQGT

SEQ ID NO: 166
LVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH

TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA

YKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST

ATKDTYDALHMQALPPR

RJ146 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(D8/CD8 TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 167
TCTTGTGCCGCCAGCGGCTTCACCTTCAGCAACTACGGCATG

AGCTGGGTCCGACAGGCCCCTGGAAAAGCCCTGGAATGGAT

CGGCGAGATCAACCACAGCGGCAGCACCAACTACAACCCCA

GCCTGAAGTCCAGAGTGACCATCAGCCGGGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGA

CACCGCCATCTACTACTGTAGAGGCGGCTGGGTCGGATATTG

GGGCCAGGGAACACTGGTCACCGTGTCTAGTGCGGCCGCAA

CCACTACTCCGGCACCTAGGCCCCCCACTCCGGCACCGACCA

TTGCATCACAACCACTGAGTCTTAGACCTGAAGCCTGTCGAC

CCGCAGCTGGGGGGGCAGTCCACACACGGGGATTGGATTTC

GCCTGCGATATATACATTTGGGCGCCACTGGCAGGCACCTGC

GGGGTCCTGCTCTTGTCCCTTGTCATCACCCTGTACTGTAAG

AGAGGAAGAAAGAAGTTGTTGTATATTTTCAAACAGCCATT

CATGAGGCCGGTCCAGACCACTCAGGAGGAGGATGGATGCA

GTTGCCGCTTTCCGGAAGAGGAAGAGGGTGGGTGTGAACTC

CGAGTTAAATTTTCTCGCAGTGCTGATGCCCCAGCGTATAAA

CAGGGTCAGAACCAACTGTATAATGAACTCAATTTGGGGAG

GCGAGAGGAGTATGATGTATTGGACAAGCGGCGGGGGCGGG

ATCCTGAAATGGGCGGTAAGCCAAGGCGCAAGAATCCACAG

GAGGGTTTGTATAATGAGTTGCAGAAGGATAAAATGGCGGA

AGCGTATTCAGAAATCGGAATGAAGGGAGAGCGCCGCAGAG

GGAAAGGGCATGATGGCCTCTACCAAGGTTTGTCAACAGCG

ACCAAGGATACCTATGACGCACTTCATATGCAAGCACTGCC

CCCCAGG

F11 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 168
TGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCAC

CTTCAGCAACTACGGCATGAGCTGGGTCCGACAGGCTCCTG

GACAAGGCTTGGAGTGGGTGTCCTACATCAGCAATGGCGGC

GGAACCATGTACTACGCCGACAGCGTGAAGGGCAGATTCAC

CATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGA

TGAACAGCCTGAGAGCCGAGGACACCGCCACCTACTACTGT

ACAAGAGCCCCTTACTGCTCTGGCGGCGACTGCTACCCTATG

AGCACACACGAGCTGGATTCTTGGGGCCAGGGCACACTGGT

CACAGTGTCATCT

RJ147 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(F11/CD8TM/
AASGFTFSNYGMSWVRQAPGQGLEWYSYISNGGGTMYYADS

4-1BB/CD3zeta)
VKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCTRAPYCSGGD

SEQ ID NO: 169
CYPMSTHELDSWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQP

LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS

LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDK

RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

RJ147 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(F11/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 170
TCTTGTGCCGCCAGCGGCTTCACCTTCAGCAACTACGGCATG

AGCTGGGTCCGACAGGCTCCTGGACAAGGCTTGGAGTGGGT

GTCCTACATCAGCAATGGCGGCGGAACCATGTACTACGCCG

ACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGC

AAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGA

GGACACCGCCACCTACTACTGTACAAGAGCCCCTTACTGCTC

TGGCGGCGACTGCTACCCTATGAGCACACACGAGCTGGATT

CTTGGGGCCAGGGCACACTGGTCACAGTGTCATCTGCGGCC

GCAACCACTACTCCGGCACCTAGGCCCCCCACTCCGGCACC

GACCATTGCATCACAACCACTGAGTCTTAGACCTGAAGCCTG

TCGACCCGCAGCTGGGGGGGCAGTCCACACACGGGGATTGG

ATTTCGCCTGCGATATATACATTTGGGCGCCACTGGCAGGCA

CCTGCGGGGTCCTGCTCTTGTCCCTTGTCATCACCCTGTACTG

TAAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCAAACAGC

CATTCATGAGGCCGGTCCAGACCACTCAGGAGGAGGATGGA

TGCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGGGTGTGA

ACTCCGAGTTAAATTTTCTCGCAGTGCTGATGCCCCAGCGTA

TAAACAGGGTCAGAACCAACTGTATAATGAACTCAATTTGG

GGAGGCGAGAGGAGTATGATGTATTGGACAAGCGGGGGG

GCGGGATCCTGAAATGGGCGGTAAGCCAAGGCGCAAGAATC

CACAGGAGGGTTTGTATAATGAGTTGCAGAAGGATAAAATG

GCGGAAGCGTATTCAGAAATCGGAATGAAGGGAGAGCGCCG

CAGAGGGAAAGGGCATGATGGCCTCTACCAAGGTTTGTCAA

CAGCGACCAAGGATACCTATGACGCACTTCATATGCAAGCA

CTGCCCCCCAGG

C52 binder
GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCC

SEQ ID NO: 171
TGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTTAC

CCTGAGCAGCTACTGGATGAGCTGGGTCCGACAGGCCCCTG

GCAAAGGACTGGAATGGATCGGCGTGATCTACTACAGCGGC

GCCACCTACTACAACCCCAGCCTGAAGTCTCTGGTCACCATC

AGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAA

CAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTA

GAGGCGCCCTGGACTACTTCGACCTTTGGGGACAAGGCGCC

CTCGTGACAGTCTCTTCT

RJ148 CAR
MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSC

(C52/CD8TM/
AASGFTLSSYWMSWVRQAPGKGLEWIGVIYYSGATYYNPSLK

4-1BB/CD3zeta)
SLVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGALDYFDLW

SEQ ID NO: 172
GQGALVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA

DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

QGLSTATKDTYDALHMQALPPR

RJ148 CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(C52/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGAGGTGCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTG

SEQ ID NO: 173
TCTTGTGCCGCCAGCGGCTTTACCCTGAGCAGCTACTGGATG

AGCTGGGTCCGACAGGCCCCTGGCAAAGGACTGGAATGGAT

CGGCGTGATCTACTACAGCGGCGCCACCTACTACAACCCCA

GCCTGAAGTCTCTGGTCACCATCAGCCGGGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGA

CACCGCCGTGTACTATTGTGCTAGAGGCGCCCTGGACTACTT

CGACCTTTGGGGACAAGGCGCCCTCGTGACAGTCTCTTCTGC

GGCCGCAACCACTACTQCGGCACCTAGGCCCCCCACTCCGG

CACCGACCATTGCATCACAACCACTGAGTCTTAGACCTGAA

GCCTGTCGACCCGCAGCTGGGGGGGCAGTCCACACACGGGG

ATTGGATTTCGCCTGCGATATATACATTTGGGCGCCACTGGC

AGGCACCTGCGGGGTCCTGCTCTTGTCCCTTGTCATCACCCT

GTACTGTAAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCA

AACAGCCATTCATGAGGCCGGTCCAGACCACTCAGGAGGAG

GATGGATGCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGG

GTGTGAACTCCGAGTTAAATTTTCTCGCAGTGCTGATGCCCC

AGCGTATAAACAGGGTCAGAACCAACTGTATAATGAACTCA

ATTTGGGGAGGCGAGAGGAGTATGATGTATTGGACAAGCGG

CGGGGGCGGGATCCTGAAATGGGCGGTAAGCCAAGGCGCA

AGAATCCACAGGAGGGTTTGTATAATGAGTTGCAGAAGGAT

AAAATGGCGGAAGCGTATTCAGAAATCGGAATGAAGGGAG

AGCGCCGCAGAGGGAAAGGGCATGATGGCCTCTACCAAGGT

TTGTCAACAGCGACCAAGGATACCTATGACGCACTTCATATG

CAAGCACTGCCCCCCAGG

H4 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 174
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVGVYYCL

QITHWPHTFGQGTKLEIKR

H4 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 175
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTG

GGCGTGTACTACTGCCTGCAGATCACACACTGGCCCCACAC

ATTTGGCCAGGGCACCAAGCTGGAAATCAAGAGA

H4 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 176
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAMYYCARDAPDLLAVPTVIFSGYHYGADVWGQGATVT

VSS

H4 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 177
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCATGTACTATTGCG

CCAGAGATGCCCCTGACCTGCTGGCTGTGCCTACCGTGATCT

TTAGCGGCTACCACTACGGCGCTGACGTTTGGGGACAAGGC

GCCACAGTGACAGTGTCTAGT

H4LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 178
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTG

GGCGTGTACTACTGCCTGCAGATCACACACTGGCCCCACAC

ATTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGTGGCG

GAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTCAA

GTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCTGGC

GGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACCTTC

AGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGGCAA

AGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCAGCA

CCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACCATC

AGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAA

CTCCCTGAGAGCCGAGGACACCGCCATGTACTATTGCGCCA

GAGATGCCCCTGACCTGCTGGCTGTGCCTACCGTGATCTTTA

GCGGCTACCACTACGGCGCTGACGTTTGGGGACAAGGCGCC

ACAGTGACAGTGTCTAGT

H4HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 179
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCATGTACTATTGCG

CCAGAGATGCCCCTGACCTGCTGGCTGTGCCTACCGTGATCT

TTAGCGGCTACCACTACGGCGCTGACGTTTGGGGACAAGGC

GCCACAGTGACAGTGTCTAGTGGTGGCGGAGGATCTGGCGG

AGGTGGAAGCGGCGGAGGCGGATCTGACATCCAGATGACAC

AGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTG

ACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCT

GAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGC

TGATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTA

GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCA

TATCTAGCCTGCAGCCTGAGGACGTGGGCGTGTACTACTGCC

TGCAGATCACACACTGGCCCCACACATTTGGCCAGGGCACC

AAGCTGGAAATCAAGAGA

RJ149LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(H4LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDVGVYYCLQITHWPHTFGQGTKLEIKRGGG

SEQ ID NO: 180
GSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSD

YYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNA

KNSLYLQMNSLRAEDTAMYYCARDAPDLLAVPTVIFSGYHYG

ADVWGQGATVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEAC

RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK

RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK

FSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM

GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH

DGLYQGLSTATKDTYDALHMQALPPR

RJ149LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(H4LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 181
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGACGTGGGCGTGTACTACTGCCTG

CAGATCACACACTGGCCCCACACATTTGGCCAGGGCACCAA

GCTGGAAATCAAGAGAGGTGGCGGAGGATCTGGCGGAGGTG

GAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTTGAATCT

GGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTGAG

CTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATGAG

CTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGTGT

CCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGATA

GCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAG

AACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGA

CACCGCCATGTACTATTGCGCCAGAGATGCCCCTGACCTGCT

GGCTGTGCCTACCGTGATCTTTAGCGGCTACCACTACGGCGC

TGACGTTTGGGGACAAGGCGCCACAGTGACAGTGTCTAGTG

CGGCCGCAACCACTACTCCGGCACCTAGGCCCCCCACTCCG

GCACCGACCATTGCATCACAACCACTGAGTCTTAGACCTGA

AGCCTGTCGACCCGCAGCTGGGGGGGCAGTCCACACACGGG

GATTGGATTTCGCCTGCGATATATACATTTGGGCGCCACTGG

CAGGCACCTGCGGGGTCCTGCTCTTGTCCCTTGTCATCACCC

TGTACTGTAAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCA

AACAGCCATTCATGAGGCCGGTCCAGACCACTCAGGAGGAG

GATGGATGCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGG

GTGTGAACTCCGAGTTAAATTTTCTCGCAGTGCTGATGCCCC

AGCGTATAAACAGGGTCAGAACCAACTGTATAATGAACTCA

ATTTGGGGAGGCGAGAGGAGTATGATGTATTGGACAAGCGG

CGGGGGGGGATCCTGAAATGGGCGGTAAGCCAAGGCGCA

AGAATCCACAGGAGGGTTTGTATAATGAGTTGCAGAAGGAT

AAAATGGCGGAAGCGTATTCAGAAATCGGAATGAAGGGAG

AGCGCCGCAGAGGGAAAGGGCATGATGGCCTCTACCAAGGT

TTGTCAACAGCGACCAAGGATACCTATGACGCACTTCATATG

CAAGCACTGCCCCCCAGG

RJ149HL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(H4HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDAPDLLAVP

SEQ ID NO: 182
TVIFSGYHYGADVWGQGATVTVSSGGGGSGGGGSGGGGSDIQ

MTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL

IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVGVYYCLQIT

HWPHTFGQGTKLEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEA

CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV

KFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE

MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

HDGLYQGLSTATKDTYDALHMQALPPR

RJ149HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(H4HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 183
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACCGCCATGTACTATTGCGCCAGAGATGCCCCTGACCTG

CTGGCTGTGCCTACCGTGATCTTTAGCGGCTACCACTACGGC

GCTGACGTTTGGGGACAAGGCGCCACAGTGACAGTGTCTAG

TGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGC

GGATCTGACATCCAGATGACACAGAGCCCTAGCAGCCTGTC

TGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCA

GCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAG

CCCGGCAAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCT

CTGCAGTCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCT

GGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAG

GACGTGGGCGTGTACTACTGCCTGCAGATCACACACTGGCC

CCACACATTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAG

CGGCCGCAACCACTACTCCGGCACCTAGGCCCCCCACTCCG

GCACCGACCATTGCATCACAACCACTGAGTCTTAGACCTGA

AGCCTGTCGACCCGCAGCTGGGGGGGCAGTCCACACACGGG

GATTGGATTTCGCCTGCGATATATACATTTGGGCGCCACTGG

CAGGCACCTGCGGGGTCCTGCTCTTGTCCCTTGTCATCACCC

TGTACTGTAAGAGAGGAAGAAAGAAGTTGTTGTATATTTTCA

AACAGCCATTCATGAGGCCGGTCCAGACCACTCAGGAGGAG

GATGGATGCAGTTGCCGCTTTCCGGAAGAGGAAGAGGGTGG

GTGTGAACTCCGAGTTAAATTTTCTCGCAGTGCTGATGCCCC

AGCGTATAAACAGGGTCAGAACCAACTGTATAATGAACTCA

ATTTGGGGAGGCGAGAGGAGTATGATGTATTGGACAAGCGG

CGGGGGCGGGATCCTGAAATGGGCGGTAAGCCAAGGCGCA

AGAATCCACAGGAGGGTTTGTATAATGAGTTGCAGAAGGAT

AAAATGGCGGAAGCGTATTCAGAAATCGGAATGAAGGGAG

AGCGCCGCAGAGGGAAAGGGCATGATGGCCTCTACCAAGGT

TTGTCAACAGCGACCAAGGATACCTATGACGCACTTCATATG

CAAGCACTGCCCCCCAGG

G6 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 184
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQ

HYGTFGQGTKLEIKR

G6 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 185
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGCTGGAAATCAAGAGA

G6 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 186
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAVYYCARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

G6 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 187
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGT

G6LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 188
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGCTGGAAATCAAGAGAGGTGGCGGAGGATCTGGCGG

AGGTGGAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTTG

AATCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGAC

TGAGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACA

TGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGG

GTGTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCC

GATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGC

CAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCG

AGGACACCGCCGTGTACTATTGCGCCAGACACAAGACCAGC

CTGAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACTAT

TGGGGCCAGGGAACACTGGTCACCGTGTCTAGT

G6HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 189
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCG

GCGGAGGCGGATCTGACATCCAGATGACACAGAGCCCTAGC

AGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTG

TAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATC

AGCAGAAGCCCGGCAAGGCCCCTAAACTGCTGATCTATGCC

GCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGATTTTCCGGC

AGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTG

CAGCCTGAGGATGCCGCCACCTACTACTGTCAGCACTACGG

CACATTTGGCCAGGGCACCAAGCTGGAAATCAAGAGA

RJ150LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(G6LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDAATYYCQHYGTFGQGTKLEIKRGGGGSGG

SEQ ID NO: 190
GGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS

WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSL

YLQMNSLRAEDTAVYYCARHKTSLNDYDFWSGYLLDYWGQG

TLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA

YKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST

ATKDTYDALHMQALPPR

RJ150LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(G6LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 191
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGATGCCGCCACCTACTACTGTCAG

CACTACGGCACATTTGGCCAGGGCACCAAGCTGGAAATCAA

GAGAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGA

GGCGGATCTCAAGTTCAGCTGGTTGAATCTGGCGGCGGACT

GGTTAAGCCTGGCGGTTCTCTGAGACTGAGCTGTGCCGCCTC

CGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACA

GGCCCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCAGCT

CCAGCGGCAGCACCATCTACTACGCCGATAGCGTGAAGGGC

AGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA

CCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGT

ACTATTGCGCCAGACACAAGACCAGCCTGAACGACTACGAC

TTTTGGAGCGGCTACCTGCTGGACTATTGGGGCCAGGGAAC

ACTGGTCACCGTGTCTAGTGCGGCCGCAACCACTACTCCGGC

ACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACC

ACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGG

GGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATAT

ACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCT

TGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAG

AAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTC

CAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCC

GGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTT

CTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAAC

CAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTA

TGATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGG

GCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTAT

AATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGA

AATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCAT

GATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATAC

CTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

RJ150HL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(G6HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHKTSLNDYD

SEQ ID NO: 192
FWSGYLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQS

PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA

SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHYGTFG

QGTKLEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA

PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

RJ150HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(G6HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 193
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACCGCCGTGTACTATTGCGCCAGACACAAGACCAGCCT

GAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACTATTG

GGGCCAGGGAACACTGGTCACCGTGTCTAGTGGTGGCGGAG

GATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGACATC

CAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGG

AGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCA

GCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCC

CCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAGTCTGGC

GTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACCGACTTC

ACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCCGCCACC

TACTACTGTCAGCACTACGGCACATTTGGCCAGGGCACCAA

GCTGGAAATCAAGAGAGCGGCCGCAACCACTACTCCGGCAC

CTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACCAC

TGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGG

GCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATATAC

ATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTG

TCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAA

GTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCA

GACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGG

AAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCT

CGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCA

ACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATG

ATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGGGC

GGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAA

TGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAA

TCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGA

TGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCT

ATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

C9 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 194
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQ

QRSNWPPTFGQGTKLEIKR

C9 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 195
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTC

GCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCTCCTAC

CTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGA

C9 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 196
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAKYYCATAVFSLGPEGYYYYMDVWGKGTTVTVSS

C9 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 197
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGATACCGCCAAGTACTACTGTG

CCACCGCCGTGTTTTCTCTGGGCCCTGAGGGCTACTACTACT

ATATGGACGTGTGGGGCAAGGGCACCACCGTGACAGTTTCT

TCT

C9LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 198
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTC

GCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCTCCTAC

CTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGTGGCG

GAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTCAA

GTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCTGGC

GGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACCTTC

AGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGGCAA

AGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCAGCA

CCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACCATC

AGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAA

CTCCCTGAGAGCCGAGGATACCGCCAAGTACTACTGTGCCA

CCGCCGTGTTTTCTCTGGGCCCTGAGGGCTACTACTACTATA

TGGACGTGTGGGGCAAGGGCACCACCGTGACAGTTTCTTCT

C9HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 199
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGATACCGCCAAGTACTACTGTG

CCACCGCCGTGTTTTCTCTGGGCCCTGAGGGCTACTACTACT

ATATGGACGTGTGGGGCAAGGGCACCACCGTGACAGTTTCT

TCTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGG

CGGATCTGACATCCAGATGACACAGAGCCCTAGCAGCCTGT

CTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCC

AGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAA

GCCCGGCAAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTC

TCTGCAGTCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTC

TGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGA

GGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGC

CTCCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGA

RJ151LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(C9LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDFAVYYCQQRSNWPPTFGQGTKLEIKRGGG

SEQ ID NO: 200
GSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSD

YYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNA

KNSLYLQMNSLRAEDTAKYYCATAVFSLGPEGYYYYMDVWG

KGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA

PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

RJ151LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(C9LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 201
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGACTTCGCCGTGTACTACTGCCAG

CAGAGAAGCAACTGGCCTCCTACCTTTGGCCAGGGCACCAA

GCTGGAAATCAAGAGAGGTGGCGGAGGATCTGGCGGAGGTG

GAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTTGAATCT

GGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTGAG

CTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATGAG

CTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGTGT

CCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGATA

GCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAG

AACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGA

TACCGCCAAGTACTACTGTGCCACCGCCGTGTTTTCTCTGGG

CCCTGAGGGCTACTACTACTATATGGACGTGTGGGGCAAGG

GCACCACCGTGACAGTTTCTTCTGCGGCCGCAACCACTACTC

CGGCACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCAC

AACCACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCT

GGGGGGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGA

TATATACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCT

GCTCTTGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAG

AAAGAAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCC

GGTCCAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCT

TTCCGGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAA

TTTTCTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAG

AACCAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGA

GTATGATGTATTGGACAAGCGGCGGGGGGGGGATCCTGAAA

TGGGCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTG

TATAATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTC

AGAAATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGG

CATGATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGA

TACCTATGACGCACTTCATATGCAAGCACTGCCCCCCAGGT

RJ151HL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(C9HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAKYYCATAVFSLGPEGY

SEQ ID NO: 202
YYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS

LSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQRSNWPPTF

GQGTKLEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA

DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

QGLSTATKDTYDALHMQALPPR

RJ151HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(C9HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 203
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GATACCGCCAAGTACTACTGTGCCACCGCCGTGTTTTCTCTG

GGCCCTGAGGGCTACTACTACTATATGGACGTGTGGGGCAA

GGGCACCACCGTGACAGTTTCTTCTGGTGGCGGAGGATCTGG

CGGAGGTGGAAGCGGCGGAGGCGGATCTGACATCCAGATGA

CACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGA

GTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTA

CCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAAC

TGCTGATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCAT

CTAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGA

CCATATCTAGCCTGCAGCCTGAGGACTTCGCCGTGTACTACT

GCCAGCAGAGAAGCAACTGGCCTCCTACCTTTGGCCAGGGC

ACCAAGCTGGAAATCAAGAGAGCGGCCGCAACCACTACTCC

GGCACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCAC

AACCACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCT

GGGGGGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGA

TATATACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCT

GCTCTTGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAG

AAAGAAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCC

GGTCCAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCT

TTCCGGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAA

TTTTCTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAG

AACCAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGA

GTATGATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAA

TGGGCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTG

TATAATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTC

AGAAATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGG

CATGATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGA

TACCTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

D7 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 204
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIAAYYCQ

HYGTFGQGTKLEIKR

D7 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 205
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATATC

GCCGCCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGCTGGAAATCAAGAGA

D7 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 206
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAVYYCARDGYPLGGGYDFWSGYYPDYWGQGTLVTVSS

D7 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 207
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTG

CCAGAGATGGCTATCCTCTTGGCGGAGGCTACGACTTTTGGA

GCGGCTACTACCCCGATTACTGGGGCCAGGGAACACTGGTC

ACAGTGTCTAGT

D7LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 208
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATATC

GCCGCCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGCTGGAAATCAAGAGAGGTGGCGGAGGATCTGGCGG

AGGTGGAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTTG

AATCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGAC

TGAGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACA

TGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGG

GTGTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCC

GATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGC

CAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCG

AGGACACCGCCGTGTACTACTGTGCCAGAGATGGCTATCCTC

TTGGCGGAGGCTACGACTTTTGGAGCGGCTACTACCCCGATT

ACTGGGGCCAGGGAACACTGGTCACAGTGTCTAGT

D7HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 209
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTG

CCAGAGATGGCTATCCTCTTGGCGGAGGCTACGACTTTTGGA

GCGGCTACTACCCCGATTACTGGGGCCAGGGAACACTGGTC

ACAGTGTCTAGTGGTGGCGGAGGATCTGGCGGAGGTGGAAG

CGGCGGAGGCGGATCTGACATCCAGATGACACAGAGCCCTA

GCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACC

TGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTA

TCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTGATCTATG

CCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGATTTTCCG

GCAGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCC

TGCAGCCTGAGGATATCGCCGCCTACTACTGTCAGCACTACG

GCACATTTGGCCAGGGCACCAAGCTGGAAATCAAGAGA

RJ152LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(D7LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDIAAYYCQHYGTFGQGTKLEIKRGGGGSGG

SEQ ID NO: 210
GGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS

WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSL

YLQMNSLRAEDTAVYYCARDGYPLGGGYDFWSGYYPDYWG

QGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA

PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

RJ152LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(D7LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 211
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGATATCGCCGCCTACTACTGTCAG

CACTACGGCACATTTGGCCAGGGCACCAAGCTGGAAATCAA

GAGAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGA

GGCGGATCTCAAGTTCAGCTGGTTGAATCTGGCGGCGGACT

GGTTAAGCCTGGCGGTTCTCTGAGACTGAGCTGTGCCGCCTC

CGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACA

GGCCCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCAGCT

CCAGCGGCAGCACCATCTACTACGCCGATAGCGTGAAGGGC

AGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA

CCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGT

ACTACTGTGCCAGAGATGGCTATCCTCTTGGCGGAGGCTACG

ACTTTTGGAGCGGCTACTACCCCGATTACTGGGGCCAGGGA

ACACTGGTCACAGTGTCTAGTGCGGCCGCAACCACTACTCCG

GCACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCACA

ACCACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTG

GGGGGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGAT

ATATACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCT

GCTCTTGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAG

AAAGAAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCC

GGTCCAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCT

TTCCGGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAA

TTTTCTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAG

AACCAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGA

GTATGATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAA

TGGGCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTG

TATAATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTC

AGAAATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGG

CATGATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGA

TACCTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

RJ152HL
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(D7HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYPLGGGY

SEQ ID NO: 212
DFWSGYYPDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT

QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY

AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIAAYYCQHYGTF

GQGTKLEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA

DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

QGLSTATKDTYDALHMQALPPR

RJ152HL
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(D7HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 213
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACCGCCGTGTACTACTGTGCCAGAGATGGCTATCCTCTT

GGCGGAGGCTACGACTTTTGGAGCGGCTACTACCCCGATTA

CTGGGGCCAGGGAACACTGGTCACAGTGTCTAGTGGTGGCG

GAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGAC

ATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGT

GGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCA

TCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAG

GCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAGTCT

GGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACCGAC

TTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATATCGCC

GCCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGCACC

AAGCTGGAAATCAAGAGAGCGGCCGCAACCACTACTCCGGC

ACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACC

ACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGG

GGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATAT

ACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCT

TGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAG

AAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTC

CAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCC

GGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTT

CTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAAC

CAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTA

TGATGTATTGGACAAGCGGCGGGGGGGGGATCCTGAAATGG

GCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTAT

AATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGA

AATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCAT

GATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATAC

CTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

B7 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 214
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQ

HYGTFGQGTKVEIKR

B7 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 215
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTC

GCCGTGTACTACTGTCAGCACTACGGCACCTTTGGCCAGGGC

ACCAAGGTGGAAATCAAGAGA

B7 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 216
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAMYYCARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

B7 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 217
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACAGCCATGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGT

B7LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 218
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTC

GCCGTGTACTACTGTCAGCACTACGGCACCTTTGGCCAGGGC

ACCAAGGIGGAAATCAAGAGAGGTGGCGGAGGATCTGGCG

GAGGTGGAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTT

GAATCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAG

ACTGAGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTA

CATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAAT

GGGTGTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACG

CCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAAC

GCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGC

CGAGGACACAGCCATGTACTATTGCGCCAGACACAAGACCA

GCCTGAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACT

ATTGGGGCCAGGGAACACTGGTCACCGTGTCTAGT

B7HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 219
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACAGCCATGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCG

GCGGAGGCGGATCTGACATCCAGATGACACAGAGCCCTAGC

AGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTG

TAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATC

AGCAGAAGCCCGGCAAGGCCCCTAAACTGCTGATCTATGCC

GCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGATTTTCCGGC

AGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTG

CAGCCTGAGGACTTCGCCGTGTACTACTGTCAGCACTACGGC

ACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGAGA

RJ153LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(B7LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDFAVYYCQHYGTFGQGTKVEIKRGGGGSG

SEQ ID NO: 220
GGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY

MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAMYYCARHKTSLNDYDFWSGYLLDYWG

QGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA

PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

RJ153LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(B7LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 221
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGACTTCGCCGTGTACTACTGTCAG

CACTACGGCACCTTTGGCCAGGGCACCAAGGTGGAAATCAA

GAGAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGA

GGCGGATCTCAAGTTCAGCTGGTTGAATCTGGCGGCGGACT

GGTTAAGCCTGGCGGTTCTCTGAGACTGAGCTGTGCCGCCTC

CGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACA

GGCCCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCAGCT

CCAGCGGCAGCACCATCTACTACGCCGATAGCGTGAAGGGC

AGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA

CCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCATGT

ACTATTGCGCCAGACACAAGACCAGCCTGAACGACTACGAC

TTTTGGAGCGGCTACCTGCTGGACTATTGGGGCCAGGGAAC

ACTGGTCACCGTGTCTAGTGCGGCCGCAACCACTACTQCGGC

ACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACC

ACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGG

GGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATAT

ACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCT

TGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAG

AAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTC

CAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCC

GGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTT

CTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAAC

CAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTA

TGATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGG

GCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTAT

AATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGA

AATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCAT

GATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATAC

CTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

RJ153HL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(B7HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARHKTSLNDYD

SEQ ID NO: 222
FWSGYLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQS

PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA

SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQHYGTFGQ

GTKVEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA

VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR

RJ153HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(B7HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 223
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACAGCCATGTACTATTGCGCCAGACACAAGACCAGCCT

GAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACTATTG

GGGCCAGGGAACACTGGTCACCGTGTCTAGTGGTGGCGGAG

GATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGACATC

CAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGG

AGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCA

GCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCC

CCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAGTCTGGC

GTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACCGACTTC

ACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCGTG

TACTACTGTCAGCACTACGGCACCTTTGGCCAGGGCACCAA

GGTGGAAATCAAGAGAGCGGCCGCAACCACTACTCCGGCAC

CTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACCAC

TGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGG

GCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATATAC

ATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTG

TCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAA

GTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCA

GACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGG

AAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCT

CGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCA

ACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATG

ATGTATTGGACAAGCGGCGGGGGGGGGATCCTGAAATGGGC

GGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAA

TGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAA

TCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGA

TGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCT

ATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

A6 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 224
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQ

RYGTFGQGTKVDIKR

A6 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 225
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGAGATACGGCACATTCGGCCAGGG

CACCAAGGTGGACATTAAGAGA

A6 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 226
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAVYYCARHKTSLNDYDFWSGYLLDYWGQGTLVTVSS

A6 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 227
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGT

A6LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 228
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGAGATACGGCACATTCGGCCAGGG

CACCAAGGTGGACATTAAGAGAGGTGGCGGAGGATCTGGCG

GAGGTGGAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTT

GAATCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAG

ACTGAGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTA

CATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAAT

GGGTGTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACG

CCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAAC

GCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGC

CGAGGACACCGCCGTGTACTATTGCGCCAGACACAAGACCA

GCCTGAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACT

ATTGGGGCCAGGGAACACTGGTCACCGTGTCTAGT

A6HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 229
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCG

CCAGACACAAGACCAGCCTGAACGACTACGACTTTTGGAGC

GGCTACCTGCTGGACTATTGGGGCCAGGGAACACTGGTCAC

CGTGTCTAGTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCG

GCGGAGGCGGATCTGACATCCAGATGACACAGAGCCCTAGC

AGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTG

TAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATC

AGCAGAAGCCCGGCAAGGCCCCTAAACTGCTGATCTATGCC

GCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGATTTTCCGGC

AGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTG

CAGCCTGAGGATGCCGCCACCTACTACTGTCAGAGATACGG

CACATTCGGCCAGGGCACCAAGGTGGACATTAAGAGA

RJ154LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(A6LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDAATYYCQRYGTFGQGTKVDIKRGGGGSG

SEQ ID NO: 230
GGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY

MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARHKTSLNDYDFWSGYLLDYWG

QGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA

PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

RJ154LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(A6LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 231
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGATGCCGCCACCTACTACTGTCAG

AGATACGGCACATTCGGCCAGGGCACCAAGGTGGACATTAA

GAGAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGA

GGCGGATCTCAAGTTCAGCTGGTTGAATCTGGCGGCGGACT

GGTTAAGCCTGGCGGTTCTCTGAGACTGAGCTGTGCCGCCTC

CGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACA

GGCCCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCAGCT

CCAGCGGCAGCACCATCTACTACGCCGATAGCGTGAAGGGC

AGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA

CCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGT

ACTATTGCGCCAGACACAAGACCAGCCTGAACGACTACGAC

TTTTGGAGCGGCTACCTGCTGGACTATTGGGGCCAGGGAAC

ACTGGTCACCGTGTCTAGTGCGGCCGCAACCACTACTCCGGC

ACCTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACC

ACTGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGG

GGGCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATAT

ACATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCT

TGTCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAG

AAGTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTC

CAGACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCC

GGAAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTT

CTCGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAAC

CAACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTA

TGATGTATTGGACAAGCGGCGGGGGGGGGATCCTGAAATGG

GCGGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTAT

AATGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGA

AATCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCAT

GATGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATAC

CTATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

RJ154HL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(A6HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHKTSLNDYD

SEQ ID NO: 232
FWSGYLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQS

PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA

SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQRYGTFGQ

GTKVDIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA

VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR

RJ154HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(A6HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 233
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGQCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACCGCCGTGTACTATTGCGCCAGACACAAGACCAGCCT

GAACGACTACGACTTTTGGAGCGGCTACCTGCTGGACTATTG

GGGCCAGGGAACACTGGTCACCGTGTCTAGTGGTGGCGGAG

GATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGACATC

CAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGG

AGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCA

GCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCC

CCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAGTCTGGC

GTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACCGACTTC

ACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCCGCCACC

TACTACTGTCAGAGATACGGCACATTCGGCCAGGGCACCAA

GGTGGACATTAAGAGAGCGGCCGCAACCACTACTCCGGCAC

CTAGGCCCCCCACTCCGGCACCGACCATTGCATCACAACCAC

TGAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGG

GCAGTCCACACACGGGGATTGGATTTCGCCTGCGATATATAC

ATTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTG

TCCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAA

GTTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCA

GACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGG

AAGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCT

CGCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCA

ACTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATG

ATGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGGGC

GGTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAA

TGAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAA

TCGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGA

TGGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCT

ATGACGCACTTCATATGCAAGCACTGCCCCCCAGG

C1 L chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

SEQ ID NO: 234
KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQ

HYGTFGQGTKVEIKR

C1 L chain
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 235
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGGTGGAAATCAAGAGA

C1 H chain
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGK

SEQ ID NO: 236
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAVYYCARHAPHKHYDFWSGYYPDYWGQGTLVTVSS

C1 H chain
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 237
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTG

CCAGACACGCCCCTCACAAGCACTACGACTTTTGGAGCGGC

TACTACCCCGACTATTGGGGCCAGGGAACACTGGTCACAGT

GTCCTCT

C1LH binder
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAG

SEQ ID NO: 238
CGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGA

GCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

AAGGCCCCTAAACTGCTGATCTATGCCGCCAGCTCTCTGCAG

TCTGGCGTGCCATCTAGATTTTCCGGCAGCGGCTCTGGCACC

GACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGATGCC

GCCACCTACTACTGTCAGCACTACGGCACATTTGGCCAGGGC

ACCAAGGTGGAAATCAAGAGAGGTGGCGGAGGATCTGGCG

GAGGTGGAAGCGGCGGAGGCGGATCTCAAGTTCAGCTGGTT

GAATCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAG

ACTGAGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTA

CATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAAT

GGGTGTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACG

CCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAAC

GCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGC

CGAGGACACCGCCGTGTACTATTGTGCCAGACACGCCCCTC

ACAAGCACTACGACTTTTGGAGCGGCTACTACCCCGACTATT

GGGGCCAGGGAACACTGGTCACAGTGTCCTCT

C1HL binder
CAAGTTCAGCTGGTTGAATCTGGCGGCGGACTGGTTAAGCCT

SEQ ID NO: 239
GGCGGTTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACC

TTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCTGG

CAAAGGCCTGGAATGGGTGTCCTACATCAGCTCCAGCGGCA

GCACCATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTG

CCAGACACGCCCCTCACAAGCACTACGACTTTTGGAGCGGC

TACTACCCCGACTATTGGGGCCAGGGAACACTGGTCACAGT

GTCCTCTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCG

GAGGQGGATCTGACATCCAGATGACACAGAGCCCTAGCAGC

CTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAG

AGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGC

AGAAGCCCGGCAAGGCCCCTAAACTGCTGATCTATGCCGCC

AGCTCTCTGCAGTCTGGCGTGCCATCTAGATTTTCCGGCAGC

GGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTGCAG

CCTGAGGATGCCGCCACCTACTACTGTCAGCACTACGGCAC

ATTTGGCCAGGGCACCAAGGTGGAAATCAAGAGA

RJ155LH CAR
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITC

(C1LH/CD8TM/
RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

4-1BB/CD3zeta)
GTDFTLTISSLQPEDAATYYCQHYGTFGQGTKVEIKRGGGGSG

SEQ ID NO: 240
GGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY

MSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARHAPHKHYDFWSGYYPDYWGQ

GTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA

VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR

RJ155LH CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(C1LH/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTGACATCCAGATGACACAG

4-1BB/CD3zeta)
AGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGAC

SEQ ID NO: 241
CATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGA

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAACTGCTG

ATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTGCCATCTAGA

TTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATA

TCTAGCCTGCAGCCTGAGGATGCCGCCACCTACTACTGTCAG

CACTACGGCACATTTGGCCAGGGCACCAAGGTGGAAATCAA

GAGAGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGA

GGCGGATCTCAAGTTCAGCTGGTTGAATCTGGCGGCGGACT

GGTTAAGCCTGGCGGTTCTCTGAGACTGAGCTGTGCCGCCTC

CGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACA

GGCCCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCAGCT

CCAGCGGCAGCACCATCTACTACGCCGATAGCGTGAAGGGC

AGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA

CCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGT

ACTATTGTGCCAGACACGCCCCTCACAAGCACTACGACTTTT

GGAGCGGCTACTACCCCGACTATTGGGGCCAGGGAACACTG

GTCACAGTGTCCTCTGCGGCCGCAACCACTACTCCGGCACCT

AGGCCCCCCACTCCGGCACCGACCATTGCATCACAACCACT

GAGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGGG

CAGTCCACACACGGGGATTGGATTTCGCCTGCGATATATACA

TTTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTGT

CCCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAAG

TTGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCAG

ACCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGGA

AGAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCTC

GCAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCAA

CTGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATGA

TGTATTGGACAAGCGGCGGGGGCGGGATCCTGAAATGGGCG

GTAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAAT

GAGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAAT

CGGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGAT

GGCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCTA

TGACGCACTTCATATGCAAGCACTGCCCCCCAGG

RJ15SHL CAR
MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSC

(C1HL/CD8TM/
AASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVK

4-1BB/CD3zeta)
GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHAPHKHYDF

SEQ ID NO: 242
WSGYYPDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSP

SSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAS

SLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHYGTFGQ

GTKVEIKRAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA

VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR

RJ155HL CAR
ATGCTTCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCC

(C1HL/CD8TM/
CACCCTGCCTTTCTGCTGATCCCTCAAGTTCAGCTGGTTGAA

4-1BB/CD3zeta)
TCTGGCGGCGGACTGGTTAAGCCTGGCGGTTCTCTGAGACTG

SEQ ID NO: 243
AGCTGTGCCGCCTCCGGCTTCACCTTCAGCGACTACTACATG

AGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGGT

GTCCTACATCAGCTCCAGCGGCAGCACCATCTACTACGCCGA

TAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCA

AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG

GACACCGCCGTGTACTATTGTGCCAGACACGCCCCTCACAA

GCACTACGACTTTTGGAGCGGCTACTACCCCGACTATTGGGG

CCAGGGAACACTGGTCACAGTGTCCTCTGGTGGCGGAGGAT

CTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTGACATCCAG

ATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGA

CAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCA

GCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCT

AAACTGCTGATCTATGCCGCCAGCTCTCTGCAGTCTGGCGTG

CCATCTAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACC

CTGACCATATCTAGCCTGCAGCCTGAGGATGCCGCCACCTAC

TACTGTCAGCACTACGGCACATTTGGCCAGGGCACCAAGGT

GGAAATCAAGAGAGCGGCCGCAACCACTACTCCGGCACCTA

GGCCCCCCACTCCGGCACCGACCATTGCATCACAACCACTG

AGTCTTAGACCTGAAGCCTGTCGACCCGCAGCTGGGGGGGC

AGTCCACACACGGGGATTGGATTTCGCCTGCGATATATACAT

TTGGGCGCCACTGGCAGGCACCTGCGGGGTCCTGCTCTTGTC

CCTTGTCATCACCCTGTACTGTAAGAGAGGAAGAAAGAAGT

TGTTGTATATTTTCAAACAGCCATTCATGAGGCCGGTCCAGA

CCACTCAGGAGGAGGATGGATGCAGTTGCCGCTTTCCGGAA

GAGGAAGAGGGTGGGTGTGAACTCCGAGTTAAATTTTCTCG

CAGTGCTGATGCCCCAGCGTATAAACAGGGTCAGAACCAAC

TGTATAATGAACTCAATTTGGGGAGGCGAGAGGAGTATGAT

GTATTGGACAAGCGGCGGGGGGGGGATCCTGAAATGGGCGG

TAAGCCAAGGCGCAAGAATCCACAGGAGGGTTTGTATAATG

AGTTGCAGAAGGATAAAATGGCGGAAGCGTATTCAGAAATC

GGAATGAAGGGAGAGCGCCGCAGAGGGAAAGGGCATGATG

GCCTCTACCAAGGTTTGTCAACAGCGACCAAGGATACCTAT

GACGCACTTCATATGCAAGCACTGCCCCCCAGG

EXAMPLES
Example 1-Preliminary Studies on Anti-FGFR4 CARs

Anti-FGFR4 CARs were developed. A recombinant extracellular domain for FGFR4 was used to screen a human scFv phage display library. The following svFv binders for FGFR4 were isolated, screened as scFv-Fc proteins, then cloned into a second-generation CAR lentiviral expression vector, featuring a human CD8 linker and transmembrane region, 4-1BB (CD137) and CD3-zeta chain (BBz) signaling domains: M408, M409, M412, M414, M415, M418, M418, M422, M424. Anti-FGR4 CARs were tested for anti-tumor lytic activity (FIG. 1). Briefly, CAR-T expressing anti-FGFR4 binders were tested against the CD19+/FGFR4⁺ cell line RH30_19, the CD19(−)/FGFR4⁺ line RMS559. Raji, a positive control for CD19-CAR, was not lysed by FGFR4-specific CARs. A control included a murine MAb-derived binder BT53-CAR; an FMC63-based CD19-specific CAR and untransduced activated T cells (UTD) served as positive and negative controls.

In some vectors the CAR sequence was followed by a T2A ribosomal skip sequence and a truncated human EGFR, to facilitate detection of T cell transductants (Wang X et al., 2011, Blood, 118:1255-63). When expressed as scFv-Fc fusion proteins, they bound FGFR4 on the surface of the following RMS tumor cell lines by flow cytometry: RMS559, R14, RH30, as well as lines including: HEK-293T expressing FGFR4, and RH30 transduced to express CD19 (RH30_19). Calculated Kd from SPR analysis (Biacore) using immobilized FGFR4 for M408, M409, M410, M414, M417 scFv-Fc and mouse BT53 (scFv from a mouse anti-FGFR4 antibody that should be effective as a CAR were 9.3 nM, 56.7 nM, 1.6 nM, 23.7 nM, 42.5 nM, and 4.2 nM, respectively. The strong activity of the CD19-CAR against the RH30_19 cell line may have been due to the higher density of the target antigen on the cell line. Using Quanti-Brite bead-based estimation of target molecule numbers per cell, the endogenously encoded FGFR4 has 19,974 targets per cell while the transgene encoded CD19 has 57,238.

A differentiator between the initial scFv candidates for CAR-T activity included in vivo activity. NSG mice were inoculated i.v. with Rh30_19 expressing FFLuc, and 3 days later following imaging, post i.p injection of luciferin, on an IVIS system were inoculated i.v. with CAR-T (Tumor+CAR), untransduced T cells (Tumor+UTD), or no T cells (Tumor Only). Weekly images were captured, with scaling changed on day 32 due to signal brightness. By day 59 all mice that did not receive FGFR4-specific CARs were sacrificed per protocol, while mice that did were alive on day 66.

When Rh30_19 cells were given i.v., followed 3 days later by CAR-T, only mice that received FGFR4-CAR were alive at day 66. (FIG. 2). This result indicated that the M410-based CAR was active in vivo. However, when a more challenging treatment model was used, where the RH30_19 RMS cell line was injected intramuscularly (i.m.), followed by CAR-T on day 3, neither FGFR4-CAR-T nor a CD19-CAR-T had an impact on tumor progression. The incomplete protection seen with FGFR4 CAR could have been due to T cell exhaustion or elimination. When peripheral blood was taken at day 11 and day 42, human T cells expressing the anti-FGFR4 CAR were readily detected by flow cytometry. On day 42, CAR+ cells accounted for 61.5% and 71.6% of the CD45+ CD8+ cells and 66.5% and 34.9% of the CD45+ CD4+ cells detected in the peripheral blood when either 3 million or 1 million CAR-T were injected i.v., respectively. Untransduced T cells accounted for 0.3 and 0.2% of the gating strategy, demonstrating antigen-dependent specificity for persistence.

As persistence per se, was not the reason for the lack of therapeutic effect, the tumor microenvironment was examined to determine if the tumor lesion contained infiltrating T cells. Tumors can be classified according to their inflammatory pathology. “Cold tumors” are non-inflamed and contain few if any T cells. “Hot” or immune-predominant tumors are abundant in lymphocytic infiltrates and are associated with better outcome and responsiveness to immune checkpoint inhibitors (Santoiemma P P, et al., 2015, Cancer Biol Ther, 16:807-20, Pages F, el al., 2018, Lancet, 391:2128-2139).

A third category of tumor-immune architecture is called “immune-excluded,” where lymphocytes are present, but appear restricted to certain regions or to the periphery of the tumor and appear to interact with stromal elements. In the i.v. Rh30_19 model, M410-based CAR, had a partially therapeutic effect (FIG. 2). Tumor progression was associated with liver metastases, while animals at takedown that did not show a luciferase signal had few or no liver metastases. Histopathology of liver metastases established two important parameters. First, tumor alone had very little stromal element in NSG mice. Second, in treated animals, a clear stroma-rich immune-excluded architecture expressing a diffuse PD-L1 staining pattern was seen.

When i.m. RH30_19 tumors were analyzed by histopathology, a similar morphology was noted in that lymphocytes were excluded or only weakly able to infiltrate the tumor (FIG. 3). Briefly, Rh30_19-bearing mice treated with M410-CAR-T were analyzed. NSG mice were injected i.m. with tumor in the hind limb and subsequently untreated, treated with M410-CAR-T, or infused with untransduced T cells. Tumors were excised, fixed, sectioned and stained for human CD8a, CD4, or with H&E. Mice that received tumor only showed no lymphocytic infiltrates. Mice that received anti-FGFR4 CAR showed an immune excluded phenotype. H&E staining revealed that tumor in CAR-treated animals had a rich stroma with broad bands of collagen, while untreated animals did not. Thus, both model systems can be used to study immune control and disease progression.

Example 2-Screening for FGFR4-Binding Polypeptides

Epitope mapping studies on FGFR4 protein demonstrated that the M410 scFv binding site was located at a site distal to the membrane, in the vicinity of the acidic box depicted in FIG. 4. It was hypothesized that creating a binder to a more membrane-proximal region would improve anti-FGFR4 CAR activity. To generate optimal anti-FGFR4 CARs, a membrane proximal domain (IgGIII) of FGFR4 was expressed as a recombinant protein and used as bait in expression library screens. Phage-display libraries were used to identify two kinds of binders. In a first set of screenings, an expression library composed of VH-only single chain binders was utilized (VH only). In a second set of screenings, a F(ab)-based library was used.

For the first set of screenings, a domain antibody (dAB) phage display library was created. The library used a single VH domain as a scaffold, and was screened for FGFR4 binders (Chen W, et al., 2008, J Mol Biol 382:779-789; Chen W, et al., 2010, Mol Immunol, 47:912-21; Chen W, et al., 2008, PNAS 105:17121-6). The phage library was cycled through at least 3 rounds of panning using plate bound recombinant extracellular domain (ECD) FGFR4 or biotin-labeled soluble antigen, and specific binders identified using soluble expression-based monoclonal ELISA (semELISA). Binders were expressed as recombinant antibodies with an HA-tag, and binding against ECD-FGFR4 quantified by ELISA was performed. For the F(ab) screening, VL-VH domain binders were identified. A set of VH-VL domain binders was derived from the VL-VH domain binders in which the VH domain preceded the VL domain in the polypeptide. Both the VL-VH domain binders and the VH-VL domain binders included a triple repeat of the amino acid sequence “GGGGS”, which linked the VH and VL chains. TABLE 3 lists certain FGFR4-binding polypeptides including: VH domain only binders identified in a screening; VL-VH domain binders identified in a screening; and VH-VL domain derived from the identified VL-VH domain binders.

Example 3-Generation of Anti-FGFR4 CARs

Polynucleotides were constructed comprising expression vectors encoding anti-FGFR4 CARs. FIG. 5 is a schematic drawing and depicts certain polynucleotides encoding: a ligand binding domain (binder), such as an FGFR4-binding polypeptide listed in TABLE 3; a CD8 transmembrane domain and linker domain; a 4-1BB domain; and a CD3 zeta domain. Some polynucleotides also included a promoter, and lentiviral vector sequences such as 5′ LTR, and 3′ SIN-LTR (SIN: self-inactivating). Some polynucleotides also included nucleic acids encoding: a leader sequence for extracellular expression; and a short ‘AAA’ linker. TABLE 7 lists anti-FGFR4 CARs containing FGFR4-binding polypeptides. Some embodiments include CARs encoded by nucleotide sequences, or comprising amino acid sequences listed in TABLE 3, TABLE 4, TABLE 5 and TABLE 6.

TABLE 7

Anti FGFR4-
FGFR4 binding

CAR
polypeptide (binder)

RJ141
E9

RJ142
A4

RJ143
E61

RJ144
A7

RJ145
D92

RJ146
D8

RJ147
F11

RJ148
C52

RJ149LH
H4-VLVH

RJ149HL
H4-VHVL

RJ150LV
G6-VLVH

RJ150HL
G6-VHVL

RJ151LH
C9-VLVH

RJ151HL
C9-VHVLH

RJ152LH
D7-VLVH

RJ152HL
D7-VHVL

RJ153LH
B7-VLVH

RJ153HL
B7-VHVL

RJ154LH
A6-VLVH

RJ154HL
A6-VHVL

RJ155LH
C1-VLVH

RJ155HL
C1-VHVL

Example 4-Cell Surface Expression of Anti-FGFR4 CARs

Polynucleotides comprising expression vectors encoding anti-FGFR4 CARs were transduced into activated T-cells.

To detect cell surface expression of the CARs, CAR T-cells were incubated with biotinylated FGFR4 protein, washed by standard centrifugation methods, incubated with streptavidin (SA)-linked-PE (phycoerythrin), washed again, and analyzed by flow cytometry. Controls included: activated T cells analyzed with no staining (unstained), or with exposure to both biotinylated FGFR4 and SA-PE (UTD, untransduced); and CARs including m410 and m412 binders.

Cells expressing the RJ146 CAR containing the VH-only binder ‘D8’ consistently showed the highest levels of binding between all CARs (FIG. 6). Cells expressing the RJ154HL CAR containing the ‘A6-VHVL’ binder showed the highest levels of binding between CARs containing the F(ab)-derived binders (FIG. 6, middle and lower panels). FIG. 6 also illustrates that not all the CARs containing binders has a specific anti-FGFR4 binding activity greater than control levels. For example, the RJ145 CAR containing the VH-only ‘D92’ binder, and the RJ148 CAR containing the VH-only ‘C52’ binder did not bind FGFR4 at levels substantially greater than control levels. In addition, a number of CARs containing the F(ab)-format binders also show essentially no FGFR4 binding, such as the RJ152LH CAR, and the RJI49HL CAR.

Example 5-In Vitro Cytotoxic Activity of Anti-FGFR4 CARs

Human T cells were activated in the presence of the cytokine IL-2, and then transduced with lentiviral (LV) vectors encoding CARs containing FGFR4 binders. To test cytotoxic activity of the anti-FGFR4 CAR T-cells, the CAR T-cells (effector cells, ‘E’) were incubated at various ratios with Rh30 cells (target cells, ‘T’) which express FGFR and also express a luciferase reporter protein. A Raji cell line, which did not express FGFR4, was used as a negative control. CAR T-cells that were activated but not transduced with LV (UTD) were used as CAR T-cell control.

As shown in FIG. 7, at lower E:T ratios, most of the effector CAR T-cells readily lysed the target Rh30 cells, and most of cells containing each CAR showed distinct killing activity. Cells containing the RJ145 CAR and the RJ148 CAR, which each contain a single chain binder had levels of killing similar to UTD controls. Cells containing the RJ149HL CAR, RJ149LH CAR, or RJ152LH CAR which each contain F(ab) derived binder also had levels of killing similar to UTD controls.

Example 6-In Vitro Cytokine Production of Anti-FGFR4 CARs

To evaluate the activity of anti-FGFR4 CAR T-cells to express interferon gamma (IFNG), the anti-FGFR4 CAR T-cells were incubated with Rh30 cells which is a rhabdomyosarcoma cell line. Cells were cultured overnight, supernatant collected, and tested for the presence of IFNG by ELISA. Unstimulated and untransduced (UTD) T cells were included as negative controls. Levels of TNF-A were also tested. As shown in FIG. 8, anti-FGFR4 CAR T-cells containing the RJ146 CAR had significant and substantially greater levels of IFNG than any other tested CAR

TABLE 8 summarizes results from the foregoing Examples. The activities of the anti-FGFR4 CAR ‘RJ146’ which contained the VH domain only ‘D8’ binder were superior to all other tested CARs.

TABLE 8

FGFR4

binding

CAR

IFN-G
TNF-A

polypeptide

expression
Cytotoxic
expression
expression

CAR
(binder)
Screen
level
activity
level
level

RJ141
E9
VH

high
high
medium

domain

only

RJ142
A4
VH

high

medium

domain

only

RJ143
E61
VH
medium
high
high
medium

domain

only

RJ144
A7
VH
medium
high
high
medium

domain

only

RJ145
D92
VH
none

domain

only

RJ146
D8
VH
highest
highest
high
high

domain

only

RJ147
F11
VH

high

medium

domain

only

RJ148
C52
VH
none

domain

only

RJ149LH
H4-VLVH
F(ab)
none

screen

RJ149HL
H4-VHVL
—
none

RJ150LV
G6-VLVH
F(ab)
high
high
high

screen

RJ150HL
G6-VHVL
—
medium
high
high
high

RJ151LH
C9-VLVH
F(ab)
low
high
high
high

screen

RJ151HL
C9-VHVL H
—
low
high
high
high

RJ152LH
D7-VLVH
F(ab)

screen

RJ152HL
D7-VHVL
—

medium

RJ153LH
B7-VLVH
F(ab)

high

screen

RJ153HL
B7-VHVL
—
medium
high
medium
high

RJ154LH
A6-VLVH
F(ab)

medium

screen

RJ154HL
A6-VHVL
—
high
high
high
high

RJ155LH
C1-VLVH
F(ab)

medium
high

screen

RJ55SHL
C1-VHVL
—

medium
medium

The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BINDERS AND CHIMERIC ANTIGEN RECEPTORS WHICH SPECIFICALLY BIND FIBROBLAST GROWTH FACTOR RECEPTOR 4

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