Methods of treating cancer by administering bispecific CD19 / CD20 car t cells

Abstract

Methods of treating cancer by administering CD19 / CD20 bispecific chimeric antigen receptor (CAR) T cells are provided.

Description

68650W001METHODS OF TREATING CANCER BY ADMINISTERING BISPECIFIC CD19 / CD20 CART CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 719,052, filed on November 11, 2024, U.S. Provisional Patent Application No. 63 / 772,236, filed on March 14, 2025, U.S. Provisional Patent Application No. 63 / 824,813, filed on June 16, 2025, U.S. Provisional Patent Application No. 63 / 857.325, filed on August 4, 2025. U.S. Provisional Patent Application No. 63 / 882,753, filed on September 16, 2025, the contents of which are all hereby incorporated by reference in their entirety into this disclosure.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on November 11, 2025, is named 68650W001_SEQ.xml and is 24,327 bytes in size.BACKGROUND

[0003] Chimeric antigen receptors (CARs) are artificial molecules that redirect the specificity of T cells to predetermined antigens. These receptors are frequently used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral or lentiviral vectors. Using adoptive transfer, autologous T cells can be genetically modified ex vivo to express a CAR specific for a cancer cell of interest. The T cells, which can then recognize and kill the cancer cells, are reintroduced into the patient. Phase I clinical studies of this approach have shown efficacy.68650W001

[0004] The most common form of CARs are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta signaling domain, which contains 3 ITAMs. CD3-zeta may not provide a fully competent activation signal and additional costimulatory signaling is needed. For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target.

[0005] Multiple clinical trials have reported remarkable therapeutic efficacy of anti-CD19 CAR-modified T cells against both acute and chronic B-cell malignancies. However, multiple cases have also been reported of patients relapsing with the emergence of CD 19-negative leukemia or lymphoma (Maude et al. 2014). This problem of antigen escape, i.e., tumor cells evading treatment by losing the antigen targeted by the T-cell therapeutic, is addressed by this invention.BRIEF SUMMARY

[0006] CD20 and CD 19 are both pan-B-cell markers present on the vast majority of malignant B cells. An OR-gate CAR that triggers tumor killing as long as either CD20 or CD 19 is present reduces the probability of antigen escape, by requiring that tumor cells lose both antigens to escape targeting, an event that happens with a significantly lower probability than singleantigen mutations. Therefore, this invention has a strong competitive advantage compared to the conventional, single-input anti-CD19 CAR T-cell therapy.

[0007] A CD19 / CD20 chimeric antigen receptor (CAR) protein construct is provided. Also provided are nucleic acids encoding the CD19 / CD20 CAR, and methods of use. e.g. in the treatment of B cell malignancies. The CD19 / CD20 CAR of the invention is a bispecific CAR that can trigger T-cell activation upon detection of either CD 19 or CD20 (or both). It is a single molecule that confers two-input recognition capability upon human T cells engineered to stably express this CAR. The CD19 / CD20 CAR consists of the following (from N- to C-terminus): signal sequence; anti CD20 scFv; linker; anti-CD19 scFv; spacer domain; transmembrane domain; zero, one, or more cytoplasmic co-stimulatory signaling domains; CD3 zeta signaling68650W001 domain. In some aspects the spacer domain is an immunoglobulin hinge domain, including without limitation the human IgG4 hinge.

[0008] In some aspects the transmembrane domain is CD28 transmembrane domain. In some aspects the cytoplasmic co-stimulatory signaling domain is CD28 and / or 4-1 BB. In some aspect the construct further comprises T2A ribosomal skipping peptide, which can be used to link the CAR to a protein or peptide of interest, e.g. an epitope tag. In some aspects a sortable tag is included, e.g. truncated epidermal growth factor receptor (EGFRt) or fluorescent proteins, which can be used to separate T cells expressing the CAR.

[0009] In some aspects the linker joining the two scFv sequences is a rigid linker. In some aspects, a rigid linker has the sequence SEQ ID NO:1 (EAAAK)n, where n is 1, 2, 3, 4, 5, 6, etc. (SEQ ID NO: 14). In some aspects, n is 3.

[0010] In some aspects the CAR construct is packaged into a lentiviral vector, which includes, without limitation, a third- generation lentiviral vector. Primary human T cells can be lentivirally transduced to stably integrate and express the OR-gate CAR. CAR-expressing cells can be enriched by fluorescence- or magnetism-activated cell sorting and expanded by antigen stimulation or stimulation with CD3 / CD28 antibodies or antibody-coated microbeads.

[0011] In some aspects of the invention, an expression vector encoding the CD19 / CD20 CAR is provided, where the vector may be a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a plasmid, or RNA.

[0012] In some aspects, a method of killing a cancer cell in an individual is provided, comprising the step of providing to the individual a therapeutically effective amount of a therapeutic cell of the invention, including an effector cell, such as a T cell, NK cell, NKT cell, etc., for example. The individual may have a B-cell malignancy, expressing one or both of CD20 and CD 19. Any method of the invention may further comprise the step of delivering to the individual an additional cancer therapy, such as surgery, radiation, hormone therapy, chemotherapy, immunotherapy, or a combination thereof, for example.

[0013] In some aspects of the invention, a kit is provided comprising cells comprising a CD19 / CD20 CAR and / or expression vector encoding a CD19 / CD20 CAR.

[0014] In some aspects of the invention, a method of preventing antigen escape in a subject in need thereof comprising administering to the subject an effective amount of a population of cells expressing a polypeptide comprising a CD19 / CD20 chimeric antigen receptor (CAR),68650W001 wherein the CAR comprises from N- to C-terminus: an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; a (G4S)nlinker, wherein n is 4 (SEQ ID NO: 19); an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; a spacer of SEQ ID NO: 5; a transmembrane domain of SEQ ID NO: 6; a co-stimulatory domain of SEQ ID NO: 8; and a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein the cells expressing the CAR are insensitive to loss of expression of either CD 19 or CD20.

[0015] In some aspect of the invention, the population of cells is a population of autologous cells. In some aspects, the population of cells is a population of T cells, NK cells, or NKT cells. In some aspects, the population of cells produces stimulatory signals for T lymphocyte proliferation and effector function following engagement of the CAR with the target antigen.

[0016] In some aspects, the subject is a cancer patient or a patient susceptible to cancer or suspected of having cancer that may be treated. In some aspects, the subject is diagnosed with lymphoma or leukemia. In some aspects, the lymphoma is B-cell non-Hodgkin lymphomas (NHL), follicular lymphoma, transformed follicular lymphoma (tFL), transformations of indolent B-cell lymphomas (excluding Richter’s transformation), diffuse large B cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), high-grade B- cell lymphoma (HGBCL) (as used herein, HGBCL includes double-hit (translocations of MYC and BCL2) HGBCL and HGBCL not otherwise specified (HGBCL NOS)), transformed marginal zone lymphoma (tMZL), or follicular lymphoma Grade 3B (Grade 3bFL).

[0017] In some aspects, the leukemia is hairy cell leukemia, B-cell chronic lymphocytic leukemia (CLL), or non-T acute lymphoblastic leukemia (ALL).

[0018] In some aspects, the subject has relapsed or refractory DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL.

[0019] In some aspects, the subject has had at least one line, or at least two lines, or at least three lines of prior therapy. In some aspects, the subject has never been administered a CAR T cell product (is CAR T naive). In some aspects, the subject has had a prior treatment with a CAR T (is CAR T experienced).

[0020] In some aspects, after administration of the CD19 / CD20 CAR cells, the subject has a response wherein the response is a complete response, a partial response, or stable disease.68650W001

[0021] In some aspects, the response after administration of the CD19 / CD20 CAR cells has a duration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or more.

[0022] In some aspects, prior to administration of the CD19 / 20 CAR cells, the subject receives one or more agents as a premedication. In some aspects, the premedication comprises acetaminophen or diphenhydramine.

[0023] In some aspects, the method further comprises administering to the patient radiation, hormone therapy, chemotherapy, immunotherapy, or a combination thereof. In some aspects, the subject may receive one or more administrations of the population of cells.

[0024] In some aspects, the population of cells are administered at the tumor site or intravenously.

[0025] In some aspects, the population of cells may be delivered at the same time or at different times as another type of cancer therapy.

[0026] In some aspects, the cells may be delivered in the same or separate formulations as another type of cancer therapy.

[0027] In some aspects, the cells may be provided to the individual in separate delivery routes as another type of cancer therapy.

[0028] In some aspects the invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a population of cells expressing a polypeptide comprising a CD19 / CD20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus; an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; a (G4S)nlinker, wherein n is 4 (SEQ ID NO: 19); an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; a spacer of SEQ ID NO: 5; a transmembrane domain of SEQ ID NO: 6; a co-stimulatory domain of SEQ ID NO: 8; and a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein between about 60% and about 99% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 65% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+.6865OWDO1

[0029] In some aspects, after administration of the population of cells, the subject has a response wherein the response comprises a complete response, a partial response, or stable disease.

[0030] In some aspects, a duration of the response after administration of the population of cells is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30. 36, 42, 48, 54, 60 months or more.

[0031] In some aspects, prior to administration of the population of cells, the subject has received a lymphodepleting chemotherapy. In some aspects, the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, bendamustine, or any combination thereof.

[0032] In some aspects, the lymphodepleting chemotherapy comprises cyclophosphamide at about 500 mg / m2 and fludarabine at about 30 mg / m2 for three days.

[0033] In some aspects, the effective amount is a therapeutically effective or pharmaceutically effective amount.

[0034] In some aspects, the effective amount of cells is a dose of 1 x 108(± 20%), 2 x 108(± 20%), or 3x 108(± 20%) cells.

[0035] In some aspects, the cancer is large cell B cell lymphoma; (b) the subject has relap sed / refractory (R / R) disease that was measurable prior to a lymphodepletion; and (c) the subject has no prior CAR T-cell exposure.

[0036] In some aspects, the subject (a) has had no more than frontline therapy; (b) relapsed within 12 months of the frontline therapy; or (c) has had primary refractory disease.

[0037] In some aspects the invention provides a method of treating DLBCL, PMBCL, HGBL, Grade 3bFL, of tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co-stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 60% of the population of cells expressing the polypeptide are CD62L+.68650W001

[0038] In some aspects, at least about 80% of the population of cells expressing the polypeptide are CD62L+.

[0039] In some aspects, the bispecific CD19 / CD20 CAR T cells are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a transactivating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate, LentiBoost™, Vectofusin-1, and poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent.

[0040] In some aspects, the viral construct comprises a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE) polynucleotide. In some aspects, the viral construct does not include a polynucleotide encoding a truncated EGFR protein. In some aspects, the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

[0041] In some aspects, the invention provides a method of treating DLBCL, PMBCL, HGBL, Grade 3bFL, of tFL in a subject in need thereof, comprising administering 100 x 106(+ 20%), 200 x 106(+ 20%) or 300 x 106(+ 20%) T cells expressing a polypeptide comprising a bispecific CD19 / 20 CAR wherein the CAR comprises from N- to C -terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 60% of the population of cells expressing the polypeptide are CD62L+; wherein the subject has received at least 2 or at least 3 prior lines of therapy.68650W001

[0042] In some aspects of the methods herein, the subject is CAR-experienced and has received at least 2 or at least 3 prior lines of therapy. In some aspects of the methods herein, the subject is CAR-naive and has received at least 2 or at least 3 prior lines of therapy.

[0043] In some aspects of the methods herein, the effective amount of cells to be administered is a dose of 1 x 108(± 20%), 2 x 108(± 20%), or 3x 108(± 20%) cells.

[0044] Some aspects of the invention provide a method of treating DLBCL, PMBCL, HGBL, Grade 3bFL, of tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 60% of the population of cells expressing the polypeptide are CD62L+ and wherein the T cells expressing the polypeptide are administered to the subject no more than between 16 and 18 days from the day source T cells are collected from the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing.

[0046] FIG. 1. Schematic of bispecific. CD20-OR-CD19 CAR. The bispecific CAR is composed of (from N to C terminal): A signal sequence that directs CAR localization to the cell membrane, the CD20 scFv, a peptide linker (e.g., (G4S)n where n is 1, 3 or 4 (SEQ ID NO: 15, 16, or 19), SEQ ID NO:1 (EAAAK)l, or SEQ ID NO: 17 (EAAAK)3), the CD19 scFv, followed by a spacer (e.g., the IgG4 hinge domain), a transmembrane domain (e.g., the transmembrane domain of CD28), one or more co- stimulatory domains (e.g., the cytoplasmic domain of 4-1 BB or CD28), and the cytoplasmic domain of CD3 chain. In one aspect, to facilitate identification of CAR-expressing T cells by antibody staining, truncated epidermal68650W001 growth factor receptor (EGFRt) can be linked to the CAR via a self-cleaving peptide (e.g., T2A).

[0047] FIG. 2A-2B. OR-gate CARs but not single-input CD 19 CARs respond to Raji lymphoma cells that have undergone antigen escape. (A) CD69, CD137, and CD107a surface expression (in median fluorescence intensity; MFI) by CAR-T cells after a 24-hour coincubation with CD19 — Raji cells. (B) IFN-y, TNF-a, and IL-2 production by the CAR-T cells in (A) as measured by cytometric bead array assay. Mock: T cells that have been mock transduced and do not express CARs. CD 19 Short: single-input CD 19 CAR with IgG4 hinge as spacer. CD20 Long: single-input CD20 CAR with IgG4 hinge-CH2-CH3 as spacer. (G4S)1 (SEQ ID NO: 15), (G4S)4 (SEQ ID NO: 19), SEQ ID NO:1 (EAAAK)l, and SEQ ID NO:17 (EAAAK)3 indicates the linker sequence of CD20-OR-CD19 CARs, all of which contain the IgG4 hinge as spacer. Reported values are the mean of triplicates, with error bars indicating one standard deviation. P-values were calculated by two-tailed Student's t test; *: p<0.05; **: p<0.01.

[0048] FIG. 3. Cell lysis by single-input and OR-gate CAR-T cells after 4-hour co-incubation with wildtype (WT; CD19+ / CD20+) or CD19 — Raji (CD19- / CD20+) cells. Reported values are the mean of triplicates, with error bars indicating one standard deviation. CAR identities are as described in FIG. 2. FIG. 3 discloses “(G4S)1” as SEQ ID NO: 15, “(G4S)4” as SEQ ID NO: 19, “(EAAAK)l” as SEQ ID NO: 1 and “(EAAAK)3” as SEQ ID NO: 17.

[0049] FIG. 4A-4B. Bispecificity is not compromised CD19 detection by OR-gate CARs. CAR-T cells were co-incubated with wildtype Raji or CD19+K562 targets for 24 hours. (A) CD69, CD137, and CD107a surface expression was quantified by flow cytometry. (B) IFN-y, TNF-a, and IL-2 production was quantified by cytometric bead array assay. Reported values are the mean of triplicates, with error bars indicating one standard deviation. CAR identities are as described in FIG. 2. FIG. 4 discloses “(G4S)1” as SEQ ID NO: 15, “(G4S)4” as SEQ ID NO: 19, “(EAAAK)l” as SEQ ID NO: 1 and “(EAAAK)3” as SEQ ID NO: 17.

[0050] FIG. 5A-5E. OR-gate CARs abrogate the effects of antigen escape in vivo. (A) Tumor progression in NSG mice bearing wildtype (WT) or mixed (75% wildtype; 25% CD19-) Raji xenografts. Bioluminescence imaging was performed on days 6, 18, and 21 post tumor injection (T cells were injected on day 7). (B) Survival of mice bearing WT or mixed Raji tumor xenografts and treated with T cells expressing no CAR or the single-input CD 19 CAR.68650W001Results indicate single-input CD19 CAR is able to significantly extend the survival of animals engrafted with WT Raji tumors. (C) Survival of mice bearing WT Raji tumor xenografts and treated with T cells expressing the single-input CD 19 CAR or OR-gate CARs. Results indicate OR-gate CARs are as efficient as single-input CD 19 CAR in targeting wildtype Raji lymphoma. (D) Survival of mice bearing mixed Raji tumor xenografts and treated with T cells expressing no CAR. the single-input CD 19 CAR, or OR-gate CARs. Results indicate only OR- gate CARs are able to significantly extend survival of animals bearing CD 19 — mutant tumors. (E) Survival of mice bearing WT or mixed Raji tumor xenografts and treated with T cells expressing OR-gate CARs. Results indicate OR-gate CARs are equally efficient against WT and CD19 — mutant Raji tumors, thus rendering the T cells insensitive to antigen loss by target cells. N=5 in all test groups. P-values were calculated by log-rank test analysis; n.s.: not significant (p>0.1); *: p<0.1; **: p<0.05. CAR identities are as described in FIG. 2. FIG. 5 discloses “(G4S)4” as SEQ ID NO: 19 and “(EAAAK)3” as SEQ ID NO: 17).

[0051] FIG. 6A-6B. Representative plasmid maps of bispecific, CD20-OR-CD19 CARs. FIG. 6A discloses “(EAAAK)3” as SEQ ID NO: 17. FIG 6B discloses GGGGS (SEQ ID NO: 15) x 4 as SEQ ID NO: 19.

[0052] FIG. 7. Schematic of dual-targeting CD19 / CD20 CAR T. (A) Schematic of the dualtargeting CD 19 / 20 CAR. (B) The dual-targeting CD19 / CD20 CAR T is manufactured using a linear process starting with CD62L+ isolation from leukapheresis, followed by activation, lentiviral transduction, cell expansion and harvest (on Day 8). CD62L is expressed on naive and central memory T-cells and not expressed on T-effector cells.

[0053] FIG. 8. Swimmer Plot of Individual Patient Trajectories over Time.

[0054] FIG. 9. Robust CAR T-cell Expansion in dual-targeting CD19 / CD20 CAR T-treated CAR-T-Naive Patients. Time plots of individual PK concentrations by droplet digital PCR (ddPCR) on DNA extracted from isolated peripheral blood mononuclear cells from 16 patients.

[0055] FIG. 10. Dual-targeting CD19 / CD20 CAR T Final Drug Product Characteristics. The final drug product from clinical lots (N = 23) is predominantly a CD3-rich product (median, 98%, range, 95 to 98%) comprising CD8 (median, 41%; range, 3 -71%) and CD4 (median, 57%; range, 23 - 96%) populations. The CD3 component of the final drug product is a CD62L- rich product comprising naive and central memory populations (median, 91%; range, 82 -68650W001

[0056] FTG. 11. Schematic of 3 + 3 dose escalation followed by dose expansion for dualtargeting CD19 / CD20 CAR T: Phase 1- 2 Trial Design.

[0057] FIG. 12. Schematic of the LYL314 Phase 1 / 2 multi-cohort, multi-center study in aggressive large B-cell lymphoma (3L+, 2L CAR-naive cohorts). See Example 3. Patients with relap sed / refractory DLBCL, PMBCL, HGBCL, Grade 3BFL, and tFL, CD19 CAR T-cell therapy naive, who have had >1 line of treatment are included in the study. CD19 / CD20 screening not required for enrollment. Study objectives include overall response rate (ORR), complete response rate (CRR), duration of response, safety and tolerability, cell expansion pharmacokinetics. 'Additional response assessments occur every 3 months for 24 months. Study included two dose levels (DL1 = 100 x 106CAR T cells; DL2 = 00 x 106CAR T cells). 100 x 106CAR T cells is the recommended Phase 2 dose. Cy, cyclophosphamide; Flu, fludarabine.

[0058] FIG. 13. Swimmer plot of 3L+ CAR-Naive patient responses at noted time post LYL314 administration as described in more detail in Example 4.

[0059] FIG. 14. Early Data in 2L Demonstrate High-Risk Patients and High Overall Response Rate. Swimmer plot of 2L+ CAR-Naive patient responses at noted time post LYL314 administration as described in more detail in Example 4.

[0060] FIG. 15A-15C. FIG. 15A is a graph showing CAR-T cell expansion (LYL314 copies per |ig DNA) post LYL314 administration. FIG. 15B is a graph showing the number of B cells / jul post CAR-T administration. FIG. 15C is graph showing %CD3+ of viable cells and %CD62L of CD3+ cells in the final drug product.

[0061] FIG. 16A-16D show boxplots comparing the expression of genes and a geneset associated with T cell memory phenotype between ronde-cel CD8+CAR+ cells vs axi-cel and tisa-cel CD8+CAR+ cells. Ronde-cel is compared to four published studies (Study 1: Deng, Q. et al (2020). Nature Medicine, 26(12), 1878-1887, Study 2: Haradhvala, N. J. et al (2022). Nature Medicine, 28(9), 1848-1859. https: / / doi.org / 10.1038 / s41591-022-01959-0 Study 3: Li, X. et al. (1837. https: / / doi.Org / 10.1016 / j.ccell.2023.08.015, Study 4: Yu, X. et al. (2025). Journal for ImmunoTherapy of Cancer, 13, e011807. https: / / doi.org / 10.1136 / jitc-2025-011807. To account for differences in cell number and sequencing depth, each product's single-cell RNAseq profile was randomly resampled 100 times (with replacement), selecting 1,000 cells per iteration. The average normalized68650W001 expression across these 100 samplings was then plotted for known memory markers: CD62L (FIG. 16A), I17R (FIG. 16B), and LEF1 (FIG.16C). FIG. 16D shows the average geneset score (Jansen, C. S. et al. (2019). An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature, 576(1181), 465-470. https: / / doi.org / 10.1038 / s41586-019-1836-5) from the 100 samplings of each product, as computed by the method GSVA (Hanzelmann, S., Castelo, R„ & Guinney, J. (2013). BMC Bioinformatics, 14(1), 7. https: / / doi.org / 10.1186 / 1471-2105- 14-7), is plotted.

[0062] FIG. 17A-17D provide boxplots comparing the expression of genes and a geneset associated with an effector phenotype, in ronde-cel, axi-cel and tisa-cel (Wherry, E. J. et al. (2007). Immunity, 27(4), 670-684. https: / / doi.Org / 10.1016 / j.immuni.2007.09.006). The values are computed in the same manner as FIG. 16.

[0063] FIG. 18 shows the median (+ / - interquartile range (IQR)) ronde-cel expansion observed in peripheral blood samples from 67 different participants at various time points post ronde-cel administration. The ronde-cel expansion is shown as the amount of FMC63 copies per one pg of DNA. LLOQ denotes the lower limit of quantification for the assay (250 copies per ug gDNA) and LLOD denotes the lower limit of detection for the assay (50 copies per ug gDNA).

[0064] FIG. 19A-19B show the correlation between memory phenotypes and peak ronde-cel expansion post-infusion in patients. P values are derived from the fit of a linear model between scaled gene or geneset expression and scaled Cmax. FIG. 19A shows the con-elation between average normalized mRNA expression from FIG. 16A of IL7R of CD8+CAR+ cells to the peak expansion in the patient post infusion (Cmax). FIG. 19B shows the correlation between Cmax and the memory geneset (Caushi, J. X. et al. (2021). Nature, 596(1816), 126-132. https: / / doi.org / 10.1038 / s41586-021-03752-4) GSVA score calculated using the method described in FIG. 16B.

[0065] FIG. 20A-20C are boxplots comparing the phenotype of CD8+CAR+ T cells collected from the peripheral blood of patients 4 weeks post-infusion of CAR-T cells for DLBCL. The figures compare ronde-cel treated patients to axi-cel treated patients from Cao et al, 2025 Biorxiv. FIG. 20A shows the proportion of CD8+CAR+ T cells that have a memory phenotype as defined by the cumulative proportion of clusters with low expression of both GZMB and KLRG1. FIG. 20B depicts the pseudobulk mRNA expression of IFNy and FIG. 20C of TNF.68650W001

[0066] FTG. 21 A-21C depict results from ex vivo experiments from three PBMC samples collected from patients 56 days post-infusion of ronde-cel. The cells were co-cultured with a Nalm6 tumor cell line and then functionality of the CAR+ cells was assessed. FIG. 21 A measures proliferation by flow cytometry with Cell Trace Violet (CTV) dye 5 days after coculture of PBMCs with Nalm6 cells at Effector:Target (CAR:Tumor) ratio of 1:1. The percentage of cells proliferating is shown for both CAR+ and CAR- cells (CAR positivity assessed by anti-G4S antibody through flow cytometry). FIG. 2 IB depicts curves showing the ratio of Nalm6 tumor cells to baseline until 72 hours after the start of the co-culture as measured by Incucyte. The line labeled Nalm6 alone is the control showing expansion of the tumor in the absence of PBMC containing CAR-T cells. The three non-labeled lines are the PBMC samples co-cultured with Nalm6 showing that up to at least 72 hours of co-culture the number of tumor cells is controlled by post-infusion ronde-cel. FIG. 21C shows the concentration of IFNy in the supernatant of the co-culture 3 days into the culture as measured by Meso Scale Diagnostics (MSD).DETAILED DESCRIPTION

[0067] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Definitions

[0068] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some aspects of the invention may consist of or consist essentially of one or more elements, method steps, and / or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other68650W001 method or composition described herein which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0069] The term “genetic modification” means any process that adds, deletes, alters, or disrupts an endogenous nucleotide sequence and includes, but is not limited to viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction, e.g., viral mediated gene transfer such as the use of vectors based on DNA viruses such as lentivirus, adenovirus, retroviruses, adeno-associated virus and herpes virus.

[0070] “Variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 80% sequence identity, more preferably, at least about 90% homologous by sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the reference amino acid sequence.

[0071] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors, such as natural killer cells, neutrophils, and macrophages, recognize bound antibody on a target cell and cause lysis of the target cell. ADCC activity may be assessed using methods, such as those described in U.S. Pat. No. 5,821,337.

[0072] “Effector cells” are leukocytes which express one or more constant region receptors and perform effector functions

[0073] As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocytes produced or processed by the thymus gland. Non-limiting classes of T cells include effector T cells and T helper (Th) cells (such as CD4+or CD8+T cells). In some aspects, the T cell is a Thl cell. In some aspects, the T cell is a Th2 cell. In some aspects, the T cell is a Tcl7 cell. In some aspects, the T cell is a Thl7 cell. In some aspects, the T cell is a Treg cell. In some aspects, the T cell is a tumor-infiltrating cell (TIL). It will be apparent to those skilled in the art that when a method provided herein comprises administering a T cell to a subject, the T cell can comprise multiple T cells. Accordingly, in some aspects, a T cell comprises a population of T cells. In some aspects, the population of T cells comprise a homogeneous mixture of multiple T cells (e.g., all the T cells have been modified to express recombinant polypeptides described herein, e.g.. all the T cells are of the same phenotype, such as all naive T cells). In some aspects, the population of T cells comprise a heterogeneous68650W001 mixture of multiple T cells (e.g., some of the T cells express recombinant polypeptides described herein while others do not, e.g., some of the T cells have different phenotypes, such as some of the T cells are naive T cells while others are memory T cells). Unless indicated otherwise, the term "T cell" and "population of T cells" are used interchangeably herein.

[0074] As used herein, the term "modified T cell" refers to a T cell that has been modified as described herein (z.e.. modified to express a dual-targeting CD19 / CD20 CAR as described herein). Unless indicated otherwise, the terms "modified T cell" and "population of modified T cells" are used interchangeably herein.

[0075] As used herein, the term "memory" T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or which have been stimulated, e.g., with an anti-CD3 antibody (e.g., in vitro or ex vivo). T cells having a "memory-like" phenotype upon secondary exposure, such memory T cells can reproduce to mount a faster and stronger immune response than during the primary exposure. In some aspects, memory T cells comprise central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), or any combination thereof.

[0076] As used herein, the term "stem cell-like memory T cells," "T memory stem cells," or "TSCM cells" refers to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.

[0077] As used herein, the term "central memory T cells" or "TCM cells" refers to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found within the lymph nodes and in peripheral circulation.

[0078] As used herein, the term "effector memory T cells" or "TEM cells" refers to memory T cells that express CD45RO but lack expression of CCR7 and CD62U. Because effector memory T cells lack lymph node-homing receptors (e.g., CCR7 and CD62U), these cells are typically found in peripheral circulation and in non-lymphoid tissues. In some aspects, effector memory T cells can be identified by a transcriptome analysis, e.g., using effector memory gene signatures disclosed, for example, in Sade-Feldman et al, Cell, 2018 Nov 1 ; 175(4):998- 1013.e20.68650W001

[0079] As used herein, the term "naive T cells" or "TN cells" refers to T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95. TN cells represent the most undifferentiated cell in the T cell lineage. The interaction between a TN cell and an antigen presenting cell (APC) induces differentiation of the TN cell towards an activated TEFF cell and an immune response.

[0080] In some aspects, a modified T cell population herein is comprised of naive and central memory T cells identified as CD62L+ T cells.

[0081] As used herein, the term "sternness," "stem cell-like." "stem-like," or "less- differentiated" refers to an immune cell (e.g., a T cell, an NK cell, or a TIL), that expresses markers consistent with a more naive phenotype. For example, a less differentiated T cell can express one or more marker characteristic of a TN or a TSCM cell. In some aspects, a "less- differentiated" or "stem-like" T cell expresses CD45RA, CCR7, and CD62L. In some aspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, CD62L, and TCF7. In some aspects, a "less-differentiated" or "stem-like" T cell does not express CD45RO or is CD45ROlow. In some aspects, the methods disclosed herein promote immune cells (e.g., T cells and / or NK cells) having a less-differentiated phenotype. Without being bound by any particular mechanism, in some aspects, the methods disclosed herein block, inhibit, or limit differentiation of less-differentiated immune cells (e.g., T cells and / or NK cells), resulting in an increased number of stem-like cells in culture. For example, it is generally thought that to effectively control tumors, adoptive transfer of less-differentiated immune cells, e.g., T cells and / or NK cells, with a stem cell-like memory or central memory phenotype are preferred. See Gattinoni, L., et al., J. Clin. Invest. 115:1616-1626 (2005). Gattinoni, L„ et al. Nat Med 15(7):808-814 (2009), Lynn, R.C., et al., Nature 576(7786): 293-300 (2019); Gattinoni, L„ et al. Nat Rev 12:671-684 (2012), Klebanoff, C., et al., J. Immunother 35(9):651-670 (2012) Gattinoni, L„ et al., Nat Med 17(10): 1290-1297 (2011); Gattinoni, L., et al. Nat Med 2 >, 18- 27 (2017); and Jansen et al Nature 2019, 576(7787):465-470.

[0082] Sternness is characterized by the capacity to self-renew. the multipotency, and the persistence of proliferative potential. In some aspects, sternness is characterized by a particular gene signature, e.g., a combined pattern of expression across a multitude of genes. In some aspects, the stem-like cells can be identified by a transcriptome analysis, e.g., using sternness gene signatures disclosed herein. In some aspects, the gene signature comprises one or more68650W001 genes selected from ACTN1 , DSC1 , TSHZ2, MYB, LEF1 , TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP. KRT72, STXBP1, TCEA3, FCGBP. CXCR5. GPA33. NELL2. APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, OCM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orfl3. GIPC3, WNT7A, FAM117B, BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LM07, CEP68, SDK2, MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, SORBS3, IL4R, SERPINF1, C16orf45, SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB. C16orf74. AEBP1, SNORD115-11. SNORD115-11, GRAP, and any combination thereof (see, e.g., Gattinoni et al.. Nature Medicine 17(10):1290-97 (2011) (Hanzelmann et al. 2013 BMC Bioinformatics 14:7, doi: 10.1186 / 1471-2105-14-7; (Jansen et al 2019, Nature Genetics 51:404-413)). In some aspects, the gene signature comprises one or more gene selected from NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR1, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293. ZC3H12D, MLXIP, C7orfl0, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SNORA5C, UBASH3A, FLJ43390, RP6- 213H19.1, INPP5A. PIM2. TNFRSF10D, SNRK, LOC100128288, PIGV, LOC100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD. TARBP1, STRADB, MT1F, TMEM41B, PDHX. KDM6B, LOC100288322. UXS1, LGMN. NANOS2, PYGB, RASGRP2, C14orf80, XPO6, SLC24A6, FAM113A, MRM1, FBXW8, NDUFS2, KCTD12, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011)). In some aspects, the gene signature comprises one or more gene selected from SELL, CCR7, S1PR1, KLF3, TCF7, GPR183, SC5D, FAAH2, LTB, SESN3, MAL, TSHZ2, LEF1, AP3M2, SLC2A3, ICAM2, PLAC8, SCML1, IL7R, ABLIM1, RASGRP2. TRABD2A, SATB1, ALG13, ARID5A, BACH2, PABPC1, GPCPD1, NELL2, TAF4B, FCMR, ARRDC2, Clorfl62,68650W001FAM177A1, ANKRD12, TXK, S0RL1 , AQP3, ADTRP, FXYD7, CD28, P2RY8, CRYBG1 , TNFSF8, BEX2, PGAP1, PTGER4, MAML2, BEX3, PCSK1N, INPP4B, AC119396.1, CXCR5, LINC00402. CCR4, IL6R, ZBTB10, ITGA6. ARMH1, RILPL2, FOXP1, TESPA1, YPEL5, LPAR6, CMSS1, RIPOR2, ZNF331, EMP3, GIMAP7, WDR74, RIC3, CYSLTR1, ITGB1, CD5, SAMHD1, SERINC5, and any combination thereof (see e.g., Caushi et al., Nature 596: 126-132 (2021)).

[0083] As used herein, the term "effector-like" or "effector cell-like" refers to tumor cell killing capacity and cytokine polyfunctionality, e.g.. ability of a cell to produce inflammatory cytokines and / or cytotoxic molecules. In some aspects, an effector-like cell is characterized by specific markers expressed by the cell. In some aspects, those effector-like markers comprise one or more of pSTAT5+, STAT5+. pSTAT3+, and STAT3+. In some aspects, the effectorlike marker comprises a STAT target selected from the group consisting of AKT1, AKT2, AKT3, BCL2L1, CBL, CBLB, CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2, CSF2RA. CSF2RB, CSF3, CSF3R. CSH1. CTF1. EP300, EPO, EPOR, GH1, GH2, GHR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL19, IL2, IL20, IL20RA, IL20RB, IL21, IL21R, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL26, IL2RA, IL2RB, IL2RG, IL3, IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, IL9, IL9R, IRF9, JAK1, JAK2, JAK3, LEP, LEPR, LIF, LIFR, MPL. MYC, OSM, OSMR, PIAS1, PIAS2. PIAS3, PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIK3R3, PIK3R5, PIM1, PRL, PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, S0S1, S0S2, SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4. STAM. STAM2, STAT1, STAT2. STAT3. STAT4, STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, and any combination thereof. In some aspects, the effector-like cells are characterized by a transcriptome analysis. In some aspects, the effector-like marker comprises a marker disclosed in Kaech et al., Cell 777:837-51 (2002); Tripathi et al., J. Immunology 785:2116-24 (2010); and / or Johnnidis et al., Science Immunology <5:eabe3702 (Jan. 15, 2021), each of which is incorporated by reference herein in its entirety.68650W001

[0084] In some aspects, the effector-like cells are characterized using an effector- associated gene set described in Gattinoni, L., et al., Nat Med 17(10): 1290-97 (2011). In some aspects, the gene signature for effector-like cells comprises one or more genes selected from MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2. PIP4K2A, HCN3. GTPBP1. TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, Clorf58, Cllorf63, C6orfl29, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA. CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB 1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B 1, RORA. C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMI, NAT13, TGFBI, FLJ00049, ANTXR2, NR4A3, IL12RB 1, NTNG2, RDX, MLLT4, GPRIN3, ADCY9, CD300A, SCD5, ABB, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1. RASGEF1A., GCNT1. GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A. FADS3. DLG5, APOBEC3D. TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA- DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK. NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MYO6, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3. TP53INP1, MBOAT1, GYG1. KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1,68650W001TBX21 , SLC1 A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1 , TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO. SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, Clorf21, KLRG1, TNIP3. GZMA. PRR5L, PRDM1. ST8SIA6. PLXND1. PTPRM, GFPT2. MYBL1, SLAMF7, FLJ16686,, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, KLRB 1, and any combination thereof (see, e.g., Gattinoni, L„ et al., Nat Med 17(10):1290-97 (2011).

[0085] The terms "subject," "patient," "individual," "participant," and "host," and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications.

[0086] To “treat” a disease or a disorder, such as cancer, means to take either therapeutic measures or preventative measures to lessen or abate the disease or disorder. Such treatment includes prevention, alleviation of symptoms, diminishment or stabilization of scope, and / or remission.

[0087] The term “therapeutically effective amount” refers to an amount of a compound or molecule effective to treat a disease or disorder

[0088] “Cancer” refers to cells undergoing uncontrolled cellular growth. Examples of cancer include colorectal cancer and head and neck cancer. A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.

[0089] A “cytokine” is a protein released by one cell to act on another cell as an intercellular mediator.

[0090] “Non-immunogenic” refers to a material that does not initiate, provoke or enhance an immune response where the immune response includes the adaptive and / or innate immune responses.

[0091] The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as68650W001 well as intervening sequences (introns) between individual coding segments (exons). Some genes may be developed which lack, in whole or in part, introns. Some leader sequences may enhance translation of the nucleic acid into polypeptides.

[0092] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0093] As used herein, a “vector” may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral vectors (e.g. retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilized onto solid phase particles. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector.

[0094] Enhancers are cis-acting elements of DNA, usually about from 10 to 300 by that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin by 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0095] “Receptor” means a polypeptide that is capable of specific binding to a molecule. Whereas many receptors may typically operate on the surface of a cell, some receptors may bind ligands when located inside the cell (and prior to transport to the surface) or may reside predominantly intra-cellularly and bind ligand therein.

[0096] The term “antibody” includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies and antibody fragments that may be human, mouse, humanized, chimeric, or derived from another species. A “monoclonal antibody” is an antibody68650W001 obtained from a population of substantially homogeneous antibodies that is being directed against a specific antigenic site.

[0097] “Antibody or functional fragment thereof’ means an immunoglobulin molecule that specifically binds to, or is immunologically reactive with a particular antigen or epitope, and includes both polyclonal and monoclonal antibodies. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies). The term functional antibody fragment includes antigen binding fragments of antibodies, including e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. The term scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.Dual-targeting OR gate CD19 / CD20 CARs

[0098] The CD19 / CD20 CARs disclosed herein are referred to as dual-targeting, OR-gate or bispecific. As used herein, “dual-targeting”, “OR gate” and “bispecific” CD19 / CD20 CARs are used interchangeably and refer to CARs that have an antigen binding domain that binds CD 19 and an antigen binding domain that binds CD20. Such CD19 / CD20 CARs are described, for example in US Patent No. 12,233,090. The present disclosure provides dualtargeting CD19 / CD20 CAR T cells that provide advantages over single targeting CAR T cells. CD20 and CD19 are both pan-B-cell markers present on the vast majority of malignant B cells. A dual-targ eting CAR that triggers tumor killing as long as either CD20 or CD 19 is present reduces the probability of antigen escape, by requiring that tumor cells lose both antigens to escape targeting, an event that happens with a significantly lower probability than singleantigen mutations. OR gate bispecific CAR T cells described herein specifically and distinctly recognize CD 19 and CD20 simultaneously or either CD20 or CD 19. Highly selective targeted T cell therapies are emerging as effective non-toxic modalities for the treatment of cancer. Malignancies are complex diseases where multiple elements contribute to the overall pathogenesis through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy without the68650W001 need to develop two separate cellular products for clinical use as combination therapy which is impractical due to regulatory hurdles and cost.

[0099] In one aspect, the bispecific CARs herein (e.g., the CD19 / CD20 bispecific CARs) are designed as a standard CAR such that the different components (e.g. , the extracellular targeting domain(s) (antigen binding domain(s)), transmembrane domain, costimulation domain, and intracellular signaling / activation domain) are linearly constructed as a single fusion protein. In one aspect, bispecific CD19 / CD20 CARs as described herein are depicted in FIG 7 A. In one aspect, such bispecific CD19 / CD20 CARs expressed in a T cell comprise, from N to C terminal, a CD20 scFv, a peptide linker, a CD 19 scFv, followed by a spacer (e.g., the IgG4 hinge domain), a transmembrane domain (e.g., the transmembrane domain of CD28), none or one or more co-stimulatory domains (e.g., the cytoplasmic domain of 4- IBB or CD28), and the cytoplasmic domain of CD3 C, chain. To facilitate identification of CAR-expressing T cells by antibody staining, or elimination of CAR-expressing T cells, a truncated epidermal growth factor receptor (EGFRt) can be linked to the CAR via a self-cleaving peptide (e.g., T2A). Such truncated EGFR are described for example in WO2021189008. In one aspect, a signal sequence that directs CAR localization to the cell membrane is included.Antigen binding domains

[0100] In one aspect, the CD 19 and CD20 antigen binding domains are each comprised of a single chain variable fragment (scFv) derived from an antibody. scFvs are recombinant molecules in which the variable regions of light and heavy immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example. Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference.

[0101] The length of the DNA linker used to link the VH and VL of an scFV is important for proper folding. It has been estimated that the peptide linker must span 3.5 nm (35 A) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site. Many such linkers are known in the art, for example flexible linkers comprising stretches of Gly and Ser residues.6865OWDO1

[0102] In one aspect, a linker is used between the two scFVs of a bispecific CAR, e.g., between the anti-CD20 scFV and the anti-CD19 scFV of a CD19 / CD20 bispecific CAR. The linkers used herein include, without limitation, a rigid linker. In some aspects of the invention, a rigid linker has the sequence (EAAAK)n, where n is 1, 2, 3, 4, 5, 6, etc. (see e.g., SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:17 or SEQ ID NO:18). In some aspects, n is 3. In one aspect, the linker has the sequence GSTSGGGSGGGSGGGGSS (SEQ ID NO: 23). In one aspect, the linker has the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 24).CD20

[0103] CD20 is a cell surface protein present on most B-cell neoplasms, and absent on otherwise similar appearing T-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkins disease, myeloma, and thymoma. CD20 is the target of the monoclonal antibodies (mAb) rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, which are all active agents in the treatment of all B cell lymphomas and leukemias. For the purposes of the present invention, any of these antibodies may be converted into a scFv and used in the CAR. In some aspects, the scFv is derived from Leu 16 monoclonal antibody. Exemplary anti-CD20 scFv sequences are set forth in SEQ ID NO: 3 and the CD20 scFV found within the complete CAR sequence provided in SEQ ID NO:21.

[0104] Cancers that may be treated with anti-CD20 reagents, e.g. antibodies and CARs, include without limitation B-cell lymphomas and leukemias, for example B-cell non-Hodgkin lymphomas (NHL), e.g. follicular lymphoma; hairy cell leukemia, and B-cell chronic lymphocytic leukemia (CLL). Anti-CD20 reagents are also useful in treating melanoma, e.g. targeting melanoma cancer stem cells.CD19

[0105] CD 19 expression is a hallmark of B cells. CD 19 antigen is a type I transmembrane glycoprotein belonging to the immunoglobulin Ig superfamily. CD 19 is specifically expressed in normal B cells and neoplastic B cells. It is considered a pan B-cell marker expressed68650W001 throughout B-cell development but with threefold higher expression in mature cells as compared to immature B cells. CD19 expression however, is lost in the terminally differentiated plasma cells. During lymphopoiesis, CD19 directs B-cell fate and differentiation by modulating B-cell receptor signaling. It is critically involved in establishing the optimal immune response through its roles in the antigen-independent development as well as the immunoglobulin-induced activation of B cells. CD 19 deficiency in humans and mice leads to an overall impaired humoral response with increased susceptibility to infection.

[0106] The pattern of CD 19 expression is maintained among B-cell malignancies where it is expressed in indolent and aggressive subtypes of B cell lymphomas and leukemias, including NHL, B-cell CLL, and non-T acute lymphoblastic leukemia (ALL). CD 19 is expressed in the B-cell lineage at an earlier stage compared with CD20. This fact, therefore, may provide an advantage to CD 19 targeted drugs over rituximab, especially for early B-cell neoplasms like acute lymphoblastic leukemia. Moreover, CD 19 is shown to be internalized efficiently in lymphoma tumor models with the use of different monoclonal antibodies (huB4, hBU12). Various anti-CD19 antibodies can be formatted for use in the constructs of the present invention, including without limitation huB4, which is a humanized anti-CD19 antibody. In one aspect, the anti-CD19 scFv is derived from the FMC63 antibody. Illustrative anti-CD19 scFv sequences are set forth in SEQ ID NO:4 and 22 and the CD19 scFV found within the complete CAR sequence provided in SEQ ID NO:21.Spacers

[0107] A spacer region links the membrane proximal antigen binding domain (e.g., the CD 19 scFv) of a bispecific CAR described herein to the transmembrane domain. It should be flexible enough to allow the antigen binding domain or domains to orient in different directions to facilitate antigen recognition. The spacer also separates the CARs antigen binding domains from the cell membrane such that they are at an optimal distance to bind their epitope(s). In one aspect, a spacer is the hinge region from an immunoglobulin, e.g. the hinge from any one of IgGl, IgG2a, IgG2b, IgG3, IgG4, particularly the human protein sequences. Alternatives spacers include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv68650W001 based constructs, an IgG hinge is effective. An illustrative spacer comprises or consists of the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:5).Transmembrane domain

[0108] In one aspect, a bispecific CD19 / CD20 CAR useful for the present disclosure comprises a transmembrane domain. In one aspect, a bispecific CD19 / CD20 CAR as described herein comprises a transmembrane domain (e.g., the transmembrane domain of CD28) following the spacer.

[0109] Any transmembrane domain known in the art can be used in the bispecific CARs described herein (e.g., bispecific CD19 / CD20 CARs). In some aspects, the transmembrane domain is artificial (e.g., an engineered transmembrane domain). In some aspects, the transmembrane domain is derived from a naturally occurring polypeptide. In some aspects, the transmembrane domain comprises a transmembrane domain from a naturally occurring polypeptide. Non-limiting examples of transmembrane domain include a transmembrane domain region of 0X40, CD2, CD27, NKG2D, NKG2C, CD19, CD8, CD28 or any combination thereof. In one aspect, the transmembrane domain comprises a CD28 transmembrane domain (e.g., such as that set forth in SEQ ID NO:6.

[0110] The CAR architecture may be any suitable architecture, as known in the art. In some aspects, a cytoplasmic signaling domain, such as those derived from the T cell receptor (^-chain, is employed as at least part of the chimeric receptor in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples would include, but are not limited to, endodomains from co- stimulatory molecules such as CD28, 4-1 BB, and 0X40 or the signaling components of cytokine receptors such as IL7 and IL15. In particular aspects, co-stimulatory domains are employed to enhance the activation, proliferation, and cytotoxicity of T cells produced by the CAR after antigen engagement. In some aspects, the co-stimulatory domains are CD28, 0X40, and 4- IBB and cytokines and their receptors, such as IL7 and IL15.68650W001

[0111] The CAR may be first generation, second generation, or third generation CAR, in which signaling is provided by CD3(^ together with co-stimulation provided by CD28 and a tumor necrosis factor receptor (TNFr). such as 4-1 BB or 0X40), for example.Costimulation domains

[0112] As described herein, in some aspects, a bispecific CD19 / CD20 CAR useful for the present disclosure comprises one or more costimulatory domains (e.g., second and third generation CARs). Not to be bound by any one theory, these costimulatory domains can further improve the expansion, activation, memory, persistence, and / or effector function of an immune cell engineered to express the bispecific CD19 / CD20 CAR (e.g., T cells). In some aspects, the transmembrane domain is fused to the costimulatory domain, optionally a costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, not limited to CD3(^. Non-limiting examples of costimulatory domain include interleukin-2 receptor (IL-2R), interleukin- 12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4. CD7, CD8, CD27, CD28, CD30. CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, or any combination thereof. In some aspects, the costimulatory domain comprises a 4-1BB / CD137 costimulatory domain (e.g., such as that set forth in SEQ ID NO:8.Intracellular signaling / activation domain

[0113] In some aspects, a cytoplasmic signaling domain, such as those derived from the T cell receptor i^-chain, is employed as at least part of the chimeric receptor in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen.

[0114] In some aspects, a bispecific CD19 / CD20 CAR described herein comprises an intracellular signaling domain that transduces the effector function signal upon binding of CD19 or CD20 and directs the cell expressing the cell expressing the bispecific CD19 / CD20 CAR (e.g., T cell) to perform a specialized function. Non-limiting examples of intracellular68650W001 signaling domain include an intracellular signaling domain region derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b. CD278 (“ICOS”), FcsRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain (e.g., such as that set forth in SEQ ID NO: 9).

[0115] The bispecific CARs described herein may be first generation, second generation, or third generation CAR, in which signaling is provided by CD3C together with co-stimulation provided by CD28 and a tumor necrosis factor receptor (TNFr), such as 4-1 BB or 0X40), for example.T2A peptide

[0116] T2 A peptide can be used to link a CAR as described herein to another protein or peptide to be expressed in the target cell. In one aspect, such other proteins include without limitation a sortable tag. T2A-linked multicistronic vectors can be used to express multiple proteins from a single open reading frame. The small T2A peptide sequences, when cloned between coding regions causes ribosomal skipping during translation when the ribosome fails to make a peptide bond at the 2A sequence, and then resumes translation (sometimes referred to as a “cleavage” event). Thus, use of the T2A peptide sequences allows for efficient, stoichiometric production of discrete protein products within a single vector. Various 2 A peptide sequences are known and used in the art, for example see Szymczak- Workman et al. (2012) Cold Spring Harb Protoc. 2012(2): 199-204, herein specifically incorporated by reference. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector.Vectors

[0117] One aspect of the present disclosure include cells that express an OR-gate CAR as described herein. The cell may be of any kind, including an immune cell capable of expressing68650W001 the OR-gate CAR described herein for cancer therapy or a cell, such as a bacterial cell, that harbors an expression vector that encodes the OR-gate CAR of the invention. As used herein, the terms “cell.” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

[0118] In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a eukaryotic cell that is capable of replicating a vector and / or expressing a heterologous gene encoded by a vector. A host cell can. and has been, used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid. In some aspects of the invention, a host cell is a T cell, including a cytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T cells or killer T cell); NK cells and NKT cells are also encompassed in the invention.

[0119] The cells can be autologous cells, syngeneic cells, allogeneic cells and even in some cases, xenogeneic cells. In many situations one may wish to be able to kill the modified T cells, where one wishes to terminate the treatment, the cells become neoplastic, in research where the absence of the cells after their presence is of interest, or other event. For this purpose one can provide for the expression of certain gene products in which one can kill the modified cells under controlled conditions, such as inducible suicide genes.

[0120] Expression vectors that encode the OR-gate CAR of the invention can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the construct(s). The constructs can be prepared in conventional ways, where the coding regions and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit may be isolated, where one or more mutations may be introduced using “primer repair”, ligation, in vitro mutagenesis, etc., as appropriate.68650W001The construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the T cells by any convenient means. The constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus. Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells. In one aspect, the expression vector is the lentiviral vector shown in FIG. 6B. The constructs may include viral sequences for transfection, if desired. Alternatively, the construct may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cells may be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s). The cells are then expanded and screened by virtue of a marker present in the construct. Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. The CAR T cells are preferably made using the methods disclosed in PCT / US 2023 / 071707, the disclosure of which is incorporated herein by reference.

[0121] In some instances, one may have a target site for homologous recombination, where it is desired that a construct be integrated at a particular locus. For example, one can knock-out an endogenous gene and replace it (at the same locus or elsewhere) with the vector encoded for by the construct using materials and methods as are known in the art for homologous recombination. For homologous recombination, one may use either omega or O-vectors. Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and / or amplifiable markers, promoter / enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available.

[0122] The T cells that have been modified with the construct(s) described herein are then grown in culture under selective conditions and cells that are selected as having the construct may then be expanded and further analyzed, using, for example; the polymerase chain reaction for determining the presence of the construct in the host cells. Once the modified host cells have been identified, they may then be used as planned, e.g. expanded in culture or introduced into a host organism.

[0123] In some aspects AAV, retroviral or lentiviral vectors are used to deliver the OR-gate CAR of the invention to a T cell.6865OWDO1

[0124] Adeno associated virus (AAV) is an attractive vector system for use in the cells of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture or in vivo. AAV has a broad host range for infectivity. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5.139,941 and 4,797,368, each incorporated herein by reference.

[0125] Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines.

[0126] Lenti viruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol. and env. contain other genes with regulatory or structural function.

[0127] Lenti viral vectors are well known in the art. Some examples of lenti virus include theHuman Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Vims: SIV. Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. In some aspects the lentiviral vector is a third generation vector (see, for example, Dull et al. (1998) J Virol. 72(11):8463-71). Such vectors are commercially available. 2nd generation lentiviral plasmids utilize the viral LTR promoter for gene expression, whereas 3rd-generation transfer vectors utilize a hybrid LTR promoter, see, for example Addgene for suitable vectors.

[0128] In some aspects, the viral vector is a retroviral vector, e.g., a lentiviral vector (e.g., a third or fourth generation lentiviral vector). The term "lenti virus" refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect nondividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that can be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.68650W001

[0129] Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (env) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. The virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system. Current third generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-mediated generation of a replication-competent virus. In fourth generation lentiviral vectors, the retroviral genome has been further reduced (see, e.g., TAKARA® LENTI-X™ fourth-generation packaging systems).

[0130] In some aspects, the present disclosure provides a lentiviral vector as depicted in the vector map in FIG. 6B. Thus, in one aspect, a lentiviral vector herein comprises a polynucleotide sequence encoding the bispecific CD19 / CD20 CAR as described herein. In some aspects, a viral vector described herein comprises a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE) polynucleotide. In some aspects, a viral vector construct herein does not include a polynucleotide encoding a truncated EGFR protein. In some aspects, a viral vector construct herein comprises a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE) polynucleotide and does not include a polynucleotide encoding a truncated EGFR protein.Methods of producing bispecific CAR T cells

[0131] In one aspect, the bispecific CD19 / CD20 CAR T cells herein are manufactured as described in W024031071, incorporated by reference herein in its entirety. In brief, autologous leukapheresis material is collected using procedures known in the art. On Day 1 cells are enriched for CD62L+ cells, for example using CD62L microbeads and CliniMACS Plus (Miltenyi Biotec) system. In one aspect, the leukapheresis product is washed with CliniMACS buffer (phosphate buffered saline (PBS) / ethylenediaminetetraacetic acid (EDTA) buffer supplemented with 0.5 percent HSA) using a Sepax C-Pro (Cytiva) and the cells incubated with the anti-CD62L microbeads and washed with the CliniMACS buffer.68650W001CD62L labeled cells are then enriched using CliniMACS plus system following manufacturer’s instructions. In one aspect, the resultant CD62L+ cells are resuspended in appropriate cell culture media, such as OpTmizer which may be supplemented with cytokines, such as IL2, IL7, IL15 or a combination thereof. In one aspect, the media is supplemented with 50 lU / mL human IL-2 and 0.5 ng / mL human IL- 15 (Miltenyi Biotec) and transferred to static cell culture bag (VueLife) or other appropriate bioreactor vessel such as a G-REX Gas Permeable Rapid Expansion device. The cells are stimulated with, for example TransAct Human T cell expander CD3 / CD28 (Miltenyi Biotec) at a predetermined ratio, such as a 1:35 ratio and cultured for about 24 hours at 37 degrees C / 5 percent carbon dioxide (CO2) in a humidified incubator. On Day 2, the cells are transduced with a lentiviral vector encoding the dual-targeting bispecific CD19 / CD20 CAR. In one aspect, the lentiviral vector encoding the dual-targeting CD19 / CD20 CAR is as depicted in FIG. 6B.

[0132] In one aspect, activated CD62L+cells are harvested from the cell culture vessel and reseeded at 0.5 x 106cells / mL in a new cell culture vessel. In one aspect, if recovered cell density is lower than 0.5 x 106, a concentration step using Sepax C-Pro (Cytiva) may be performed. In one aspect, lentiviral vector encoding the CD19 / CD20 CAR is added cells at a multiplicity of infection (MOI) of 5-15 and the cells with the vector are incubated for 1-14 days, supplementing fresh media as needed. In one aspect, the cells are incubated for 2-10 days, for 2-9 days, for 2-8 days, for 2-7 days, for 2-6 days.

[0133] In one aspect, on Day 6 of the process, transduced T cells are washed to remove residual TransAct using a Sepax C-Pro (Cytiva) and seeded in a fresh culture bag with media supplemented with cytokines at a density of 0.5 x 106cells / mL and incubated at 37 + 2 degrees centigrade with 5 + 1 percent CO2 and 95 percent RH (acceptable range 75 percent to 99 percent) for an additional 2 days.

[0134] In one aspect, on Day 8, cells are harvested and washed to remove media using Plasmalyte plus HSA on Sepax C-Pro (Cytiva). In one aspect, if the target cell number for infusion is not reached at Day 8, cells will be kept in culture for 2 additional days (Day 10) or 4 additional days (Day 12) with regular media feeds with fresh cytokines.

[0135] In one aspect, the washed cells are formulated with cryopreservation solution, such as CryoStor CS 10 Freezing Solution (BioLife), filled into bags / vials at predetermined cell density and volume, and, in one aspect, cryopreserved using a controlled rate freezer, such as68650W001ViaFreeze (Cytiva). In one aspect, the final formulation is 50 percent Plasmalyte with 5 percent HSA and 50 percent CS10 (5 percent dimethylsulfoxide (DMSO)). Cryopreserved products are stored in vapor phase liquid nitrogen freezer.

[0136] In one aspect of the present disclosure, the methods described herein produce a population of T cells wherein at least about 80% of the CD3+ T cells are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the population of the T cells are CD62L+. In one aspect, from about 35% to about 95% of the produced T cells are CAR+. In one aspect, from about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher of the produced T cells are CAR+.

[0137] In one aspect of the present disclosure, the methods described herein produce a population of bispecific CD19 / CD20 CAR T cells wherein from about 50% to about 80% of the CAR T cells are CD62L+. In one aspect of the present disclosure, the methods described herein produce a population of bispecific CD19 / CD20 CAR T cells wherein from about 50% to about 60%, from about 55% to about 60%, from about 50% to about 65%, from about 55% to about 65%, from about 60% to about 65%, from about 60% to about 70%, or from about 75% to about 80% of the CAR T cells are CD62L+. In one aspect of the present disclosure, the methods described herein produce a population of bispecific CD19 / CD20 CAR T cells wherein at least about 50%, 55%, 60%, 65%, or 70% of the CAR T cells are CD62L+. In one aspect, at least about 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%, or 100% of the population of the CAR T cells are CD62L+. In one aspect, from about 35% to about 95% of the produced T cells are CAR+. In one aspect, from about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher of the produced T cells are CAR+.68650W001Dosages and Dosage Regimens.

[0138] Therapeutically effective amounts or dosages are contemplated to include dosages of the bispecific CD19 / CD20 CAR T cells between about 106and about 109CAR T cells and can be more than IO10CAR T cells.

[0139] In some aspects, the bispecific CD19 / 20 CAR T cells are administered at a dose of 1 x 108(± 20%), 2 x 108(± 20%), or 3x 108(± 20%) CAR T cells. In some aspects, the bispecific CD19 / 20 CAR T cells are administered at a dose of 1 x 107(± 20%), 2 x 107(± 20%), 3x 107(± 20%), 4 x 107(± 20%), 5 x 107(± 20%), 6x 107(± 20%), 7 x 107(± 20%), 8 x 107(± 20%), or 9x 107(± 20%) CAR T cells.

[0140] In some aspects, the bispecific CD19 / 20 CAR T cells are administered at a dose of 50 x 106(± 20%), 60 x 106(± 20%), 70x 106(± 20%), 80x 106(± 20%), 90x 106(± 20%), or lOOx 106(± 20%) CAR T cells. In some aspects, the bispecific CD19 / 20 CAR T cells are administered at a dose of 125 x 106(± 20%), 150 x 106(± 20%), 175 x 106(± 20%), 200 x 106(± 20%), 225 x 106(± 20%), 250 x 106(± 20%), 275 x 106(± 20%), or 300 x 106(± 20%) CAR T cells.

[0141] In some aspects, the cells herein are formulated by washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but also 5% dextrose in water or Ringer’s lactate can be utilized. The infusion medium can be supplemented with human serum albumin.

[0142] The density of the desired cells is typically greater than 104, 105, or 106cells / ml. The clinically relevant number of immune cells (e.g., CD19 / CD20 bispecific CAR modified T cells as described herein) can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, or IO10cells. In some aspects of the present disclosure, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106 / kilogram (1O6-1O10per patient) may be administered. Treatments comprising immune cells, e.g., T cells, prepared according to the methods disclosed herein may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy.68650W001

[0143] In one aspect a subject in need thereof is provided with therapeutic T cells modified to comprise an OR-gate CAR of the present disclosure. The cells may be delivered at the same time or at different times as another type of cancer therapy. The cells may be delivered in the same or separate formulations as another type of cancer therapy. The cells may be provided to the individual in separate delivery routes as another type of cancer therapy. The cells may be delivered by injection at a tumor site or intravenously or orally, for example. Routine delivery routes for such compositions are known in the art.Lymphodepleting Agent

[0144] In one aspect, the dual-targeting CD19 / CD20 CAR T cells described herein are administered to a subject in need thereof after a lymphodepleting agent. As used herein, the term "lymphodepleting agent" refers to any agent that is capable of reducing, depleting, or eliminating a subject's endogenous lymphocytes (e.g., T cells) when administered to the subject. Any suitable lymphodepleting agents known in the art can be used with the present disclosure. See, e.g., Lickefett et al., Front Immunol 14: 1303935 (2023), which is incorporated herein by reference in its entirety. Non-limiting examples of suitable lymphodepleting agents include a fludarabine, cyclophosphamide, oxaliplatin, bendamustine, busilvex, alemtuzumab, total lymphoid irradiation (TLI), allo-647, etoposide, VP-16, cytarabine, dexamethasone, nirogacestat, or combinations thereof. In some aspects, the lymphodepleting agent comprises fludarabine and cyclophosphamide. In some aspects, the lymphodepleting agent comprises fludarabine and bendamustine. In some aspects, the lymphodepleting agent comprises fludarabine and busilvex. In some aspects, the lymphodepleting agent comprises fludarabine, cyclophosphamide, and alemtuzumab. In some aspects, the lymphodepleting agent comprises fludarabine, cyclophosphamide, and TLI. In some aspects, the lymphodepleting agent comprises fludarabine, cyclophosphamide, and allo-647. In some aspects, the lymphodepleting agent comprises fludarabine, cyclophosphamide, and etoposide. In some aspects, the lymphodepleting agent comprises fludarabine, cyclophosphamide, and VP- 16.

[0145] In one aspect, the lymphodepleting agent is administered to the subject prior to the CD19 / CD20 CAR modified T cell, such that the subject's endogenous lymphocytes (e.g., T cells) are depleted or reduced when the modified T cell is administered to the subject. For68650W001 instance, in some aspects, the modified T cell is administered to the subject at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours after the lymphodepleting agent. In some aspects, the modified T cell is administered about 12 hours to about 120 hours, about 24 hours to about 120 hours, about 36 hours to about 120 hours, about 48 hours to about 120 hours, about 60 hours to about 120 hours, about 72 hours to about 120 hours, about 84 hours to about 120 hours, about 96 hours to about 120 hours, about 108 hours to about 120 hours after the lymphodepleting agent.Methods of Treatment

[0146] By way of illustration, cancer patients or subjects susceptible to cancer or suspected of having cancer may be treated as described herein. Cancers include particularly B-cell leukemias and lymphomas. T cells modified as described herein (e.g., bispecific CD19 / CD20 CAR T cells) may be administered to the patient and retained for extended periods of time. The individual may receive one or more administrations of the cells. In some aspects, the genetically modified cells are encapsulated to inhibit immune recognition and placed at the site of the tumor. The cells may be injected at the tumor site or injected intravenously, for example.

[0147] Methods of treatment herein comprise one or more administrations of bispecific CD19 / CD20 cells in doses disclosed herein. In some aspects, methods comprise one administration of bispecific CD19 / CD20 cells. In some aspects, methods comprise two administrations of bispecific CD19 / CD20 cells. In some aspects, methods comprise three administrations of bispecific CD19 / CD20 cells. In some aspects, methods comprise four administrations bispecific CD19 / CD20 cells. In some aspects, methods comprise five administrations of bispecific CD19 / CD20 cells. In some aspects, methods comprise six administrations of bispecific CD19 / CD20 cells. In some aspects, methods comprise more than six administrations of bispecific CD19 / CD20 cells.

[0148] In some aspects, the bispecific CD19 / CD20 cells are administered according to a dosing interval (e.g., a cycle). In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered daily. In some aspects, one or more administrations the68650W001 bispecific CD19 / CD20 cells are administered weekly. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered biweekly. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered every three weeks. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered monthly. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered every two months. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered every three months. In some aspects, one or more administrations the bispecific CD19 / CD20 cells are administered every two months. In some aspects, one or more administrations of bispecific CD19 / CD20 cells are administered yearly.

[0149] In some aspects, the bispecific CD19 / CD20 cells are administered according to a dosing interval (e.g., a cycle). In some aspects, the dosing interval comprises a three-week cycle and the bispecific CD19 / CD20 cells are administered once every three weeks. In some aspects, the dosing interval is repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some aspects, the repeated dosing interval is performed over at least 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, or 5 years.

[0150] In some aspects, the bispecific CD19 / CD20 cells are administered according to individual subject responses. For example, in some aspects, the bispecific CD19 / CD20 CAR T cells are administered to a subject in need thereof and readministered if the subject responds but then later progresses. In some aspects, the bispecific CD19 / CD20 cells are readministered to a subject within 1 week, 2 weeks, or 3 weeks, of disease progression. In some aspects, the bispecific CD19 / CD20 CAR T cells are readministered to a subject within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days of disease progression. As would be understood by a person of ordinary skill in the art, the time of readministration can be determined by a clinician depending on the patient.

[0151] The immune cells, e.g., the bispecific CD19 / CD20 CAR T cells, of the present disclosure may be administered either alone, or as a pharmaceutical composition in68650W001 combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present disclosure may comprise a T cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are preferably formulated for intravenous administration. Treatment may also include one or more corticosteroid treatment, such as dexamethasone and / or methylprednisolone.

[0152] Some aspects of the present disclosure relate to a method of treating a cancer in a subject in need thereof, comprising administering a population of cells (e.g., T cells) expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (648)4 linker or a (648)1 linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co-stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% of the population of cells (e.g.. T cells) expressing the polypeptide are CD62L+.

[0153] Some aspects of the present disclosure relate to a method of treating a cancer in a subject in need thereof, comprising administering a population of cells (e.g., T cells) expressing a polypeptide comprising a CD19 / C20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (&4S)nlinker, wherein n is 1, 3, or 4 (SEQ ID NO: 15, 16, or 19); c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co-stimulatory domain of SEQ ID NO: 8; and68650W001 g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein between about 60% and about 99% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 99% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 98% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 97% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 96% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 90% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 85% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 75% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 60% and about 70% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 99% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 98% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 97% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 96% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 90% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 85% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 75% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 65% and about 70% of the68650W001 population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 75% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 75% and about 90% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 75% and about 85% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 75% and about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 80% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 80% and about 90% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 80% and about 85% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 85% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 85% and about 90% of the population of cells (e.g.. T cells) expressing the polypeptide are CD62L+. In some aspects, between about 90% and about 99% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, between about 90% and about 95% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+.

[0154] In some aspects, the dual-targeting CD19 / CD20 CAR T cells has key features designed to increase the complete response rate (CRR) and to prolong the duration of those responses. In some aspects, the feature is a bispecific CAR that targets two B-cell antigens, CD19 and CD20, thereby reducing the risk of progression due to target loss variants or low target density. In some aspects, the feature is the enrichment of long-lived naive and central memory T cells in the manufacturing process, thereby promoting in vivo persistence of CAR T cells.

[0155] In some aspects, the present disclosure provides methods of treating cancer in a subject by administering a dual-targeting CD19 / CD20 CAR T cell product wherein the cancer is selected from aggressive B-cell non-Hodgkin lymphoma (NHL), including large B cell lymphoma (LBCL), Diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B- cell lymphoma (PMBCL), diffuse large B cell lymphoma arising from follicular lymphoma (transformed follicular lymphoma [tFL]), transformations of indolent B-cell lymphomas (excluding Richter’s transformation), high-grade B-cell lymphoma (HGBCL), (as used herein,6865OWDO1HGBCL includes double-hit (translocations of MYC and BCL2) HGBCL and HGBCL not otherwise specified (HGBCL NOS)), and follicular lymphoma Grade 3B.

[0156] In some aspects, the subject has relapsed or refractory DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL. In some aspects, the subject has had at least one line, or at least two lines, or at least three lines of prior therapy. In some aspects, the subject is CAR T naive (e.g., has never been treated with a CAR T cell product). In some aspects, the subject is CAR T experienced (e.g., has been previously treated with a CAR T cell product). In some aspects, the subject has had no more than one line of prior therapy. In some aspects, the subject has had only frontline therapy. In some aspects, the subject is CAR-experienced and has received 3 or more prior lines of therapy (CAR-experienced 3rdLine+). In some aspects, the subject has relapsed or refractory disease after anti-CD20 antibody and anthracycline-containing first- line chemoimmunotherapy. First (frontline), second and third + line therapies for DLBCL, PMBCL, HGBCL, Grade 3bFL, and TFL are known in the art and are determined by clinicians treating subjects in need thereof. In some aspects, prior lines of therapy include but are not limited to any anthracycline drug, R-CHOP (one or more chemotherapy drugs cyclophosphamide, doxorubicin, vincristine, and prednisone, along with rituximab); Pola-R- CHP (adding polatuzumab vedotin monoclonal antibody, to the standard R-CHOP chemotherapy); bispecific antibodies; DA-R-EPOCH (a dose-adjusted regimen that includes rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin); R- V / MACOP-B (rituximab and various chemotherapy drugs); Zanubrutinib; anti-CD19 antibodies; anti-CD20 antibodies; radiotherapy; EOT-PET (end-of-treatment positron emission tomography); checkpoint inhibitors (e.g., pembrolizumab. ipilimumab, atezolizumab); stem cell transplant; polatuzumab; rituximab-bendamustine; CAR-T cell therapy (e.g., axicabtagene, lisocatagene); tafasitamab-lenalidomide; polatuzumab vedotin- piiq; gemcitabine and / or oxaliplatin with or without rituximab; Selinexor; epcoritamab; glofitamab; R-ICE (rituximab, ifosfamide, carboplatin, etoposide); R-DHAP (rituximab, dexamethasone, high-dose cytarabine, cisplatin); GDP (gemcitabine, dexamethasone, cisplatin); DHAP (dexamethasone, cytarabine, cisplatin); IGE(V) (ifosfamide, gemcitabine, vinorelbine); rituximab or obinutuzumab combined with CHOP-like chemotherapy or bendamustine; and chemoimmunotherapy.6865OWDO1

[0157] Some aspects of the present disclosure provide a method of treating DLBCL, PMBCL,HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, the subject achieves a CR for at least 6 months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the subject achieves a CR for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the cells expressing the bispecific CD19 / CD20 CAR are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a trans activating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate. LentiBoost™, Vectofusin-1, and poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent. In some aspects, the viral construct comprises a lentiviral vector as depicted in FIG. 6B. In some aspects the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide; or the viral construct does not include a polynucleotide encoding a truncated EGFR protein; or the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory68650W001Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

[0158] Some aspects of the present disclosure relate to a method of treating DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%. 84%. 85%, 86%, 87%. 88%. 89%, 90%, 91%. 92%. 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+ where the subject has previously received only frontline therapy. In some aspects, the subject achieves a CR for at least 6 months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the subject achieves a CR for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the cells expressing the bispecific CD19 / CD20 CAR are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a transactivating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate, LentiBoost™, Vectofusin-1, and poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent. In some aspects, the viral construct comprises a lentiviral vector as depicted in FIG. 6B. In some aspects the viral construct comprises a Woodchuck Hepatitis68650W001Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide; or the viral construct does not include a polynucleotide encoding a truncated EGFR protein; or the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

[0159] Some aspects of the present disclosure relate to a method of treating DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+ wherein the subject has previously received one prior line of therapy. In some aspects, the subject achieves a CR for at least 6 months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the subject achieves a CR for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20 or more months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the cells expressing the bispecific CD19 / CD20 CAR are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a transactivating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate, LentiBoost™, Vectofusin-1, and6865OWDO1 poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent. In some aspects, the viral construct comprises a lentiviral vector as depicted in FIG. 6B. In some aspects the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide; or the viral construct does not include a polynucleotide encoding a truncated EGFR protein; or the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

[0160] Some aspects of the present disclosure relate to a method of treating DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+ where the subject has received two prior lines of therapy. In some aspects, the subject achieves a CR for at least 6 months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the subject achieves a CR for at least 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19, 20 or more months following administration of the bispecific CD19 / CD20 CAR. In some aspects, the cells expressing the bispecific CD19 / CD20 CAR are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a transactivating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral6865OWDO1 construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate, LentiBoost™. Vectofusin-1, and poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent. In some aspects, the viral construct comprises a lentiviral vector as depicted in FIG. 6B. In some aspects the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide; or the viral construct does not include a polynucleotide encoding a truncated EGFR protein; or the viral construct comprises a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

[0161] Some aspects of the present disclosure relate to a method of treating DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+.

[0162] In some aspects of the methods of treatment, the T cells expressing the polypeptide are administered to a subject no more than between 16 and 18 days from the day the subject’s cells are collected before treatment, e.g., by apheresis (source T cells). Some aspects relate to a method of treating DLBCL, PMBCL, HGBCL, Grade 3bFL, tMZL, or tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(+ 20%) cells expressing a polypeptide comprising a bispecific CD 19 / 20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising68650W001 a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (648)4 linker or a (648)1 linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 80% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+, and wherein the T cells expressing the polypeptide are administered to a subject no more than between 16 and 18 days from the day the source cells are collected. In some aspects, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the population of cells (e.g., T cells) expressing the polypeptide are CD62L+.

[0163] In some aspects, the subject to be treated is one who has previously been treated with a prior therapy. In some aspects, the subject responded (e.g., had a complete response or partial response) to the prior therapy. In some aspects, the subject had a period of response of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or more following administration of the prior therapy.

[0164] In some aspects, the subject progressed following a period of response to the prior therapy. In some aspects, the subject receives treatment with the bispecific CD 19 / 20 CAR cells following a progression after the period of response to the prior therapy. In some aspects, the subject had stable disease upon administration of the prior therapy. In some aspects, the subject had a period of stable disease of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or more. In some aspects, the subject progressed following a period of stable disease after treatment with a prior therapy. In some aspects, the subject receives treatment with the bispecific CD19 / 20 CAR cells following progression after the period of stable disease after treatment with the prior therapy.

[0165] In some aspects, treatment with the prior therapy ceased before initiation of administration of the CD 19 / 20 CAR cells. In some aspects, treatment with the prior therapy continues after initiation of administration of the CD19 / 20 CAR cells.

[0166] In some aspects, treatment with the CD19 / 20 CAR cells results in a response (e.g., a complete response or partial response) or stable disease. In some aspects, the response after68650W001 administration of the CD 19 / 20 CAR cells persists for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 18, 24, 30, 36, 42, 48, 54, 60 months or more.

[0167] In some aspects, prior to administration of the CD19 / 20 CAR cells, the subject receives bridging therapy.

[0168] In some aspects, prior to administration of the CD19 / 20 CAR cells, the subject receives one or more agents as a premedication.

[0169] In some aspects, the premedication comprises acetaminophen or diphenhydramine. In some aspects, the subject receives acetaminophen or diphenhydramine 30 to 120 minutes prior to administration of the CD19 / 20 CAR cells. In some aspects, the premedication is administered orally or by intravenous injection.

[0170] In some aspects, the CD19 / CD20 CAR cells are administered by intravenous administration. In some aspects, the cells are administered locally. In some aspects, the CD19 / CD20 CAR cells are formulated as a solution. In one aspect, the CD19 / CD20 CAR T cell treatment consists of a single infusion of CAR-transduced autologous T cells administered intravenously after a conditioning chemotherapy regimen consisting of fludarabine 30 mg / m2 / day and cyclophosphamide 500 mg / m2 / day for CAR T naive cohort, or fludarabine 30 mg / m2 / day and cyclophosphamide 300 mg / m2 / day for CAR T experienced cohort, administered over 3 days, or bendamustine 90mg / m2 over 2 days.

[0171] In some aspects, the subject is monitored prior to administration of the dual targeting CD 19 / 20 CAR cells. Symptoms are identified and their severity is assessed. The CD 19 / 20 CAR cells as described herein may be administered alone or in combination with additional treatments, singly or multiply over time as discussed herein or known to one of skill in the art. In some aspects, the subject is monitored such that the efficacy of the treatment regimen is determined. In some aspects, a treatment regimen is modified in response to preliminary treatment outcomes, such that treatment dose or frequency or dose and frequency is altered so as to attain a desired level of subject response in light of symptom alleviation, side effect reduction, or a combination of symptom alleviation and side effect reduction.

[0172] In some aspects, the compositions further comprise an additional therapeutic agent. In some aspects, the therapeutic agent is a chemotherapeutic agent. In some aspects, the CD 19 / 20 CAR cells and the therapeutic agent are in the same formulation. In some aspects, the CD 19 / 20 CAR cells and the therapeutic agent are in different formulation. In some aspects, CD 19 / 2068650W001CAR cells described herein is used prior to the administration of the other therapeutic agent. In some aspects, CD 19 / 20 CAR cells described herein is used concurrently with the administration of the other therapeutic agent. In some aspects, CD 19 / 20 CAR cells described herein is used subsequent to the administration of the other therapeutic agent. In some aspects of the invention, methods of the present invention for clinical aspects are combined with other agents effective in the treatment of hyperproliferative disease, such as anti-cancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cancer cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, Abraxane®, Altretamine®, docetaxel, Herceptin®, methotrexate, Novantrone™, Zoladex™, Cisplatin (COOP), Carboplatin, proCarbaZine. mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16™), tamoxifen, raloxifene, estrogen receptor binding agents. Taxol®, gemcitabien. Navelbine®, famesyl-protein transferase inhibitors, transplatinum, 5 -fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing and also combinations thereof. In some aspects, a modified T cell described herein can be administered to a subject in combination with an additional agent. In some aspects, the additional agent comprises a lymphodepleting agent. Accordingly, some aspects of the present disclosure are related to a method of treating a solid tumor in a subject in need thereof, comprising administering to the subject a modified T cell68650W001 as described herein and a lymphodepleting agent, wherein the modified T cell expresses recombinant polypeptides comprising a dual-targeting CD19 / CD20 CAR.

[0173] The compositions of the present application can comprise, consist essentially of, or consist of, the components disclosed.

[0174] The pharmaceutical compositions of the disclosure (solutions, suspensions or the like), may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene-diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.Kits

[0175] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, one or more cells for use in cell therapy and / or the reagents to generate one or more cells for use in cell therapy that harbors recombinant expression vectors may be comprised in a kit. The kit components are provided in suitable container means. Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the components in close68650W001 confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0176] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular aspects of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding aspects described herein may be utilized according to the present invention.

[0177] Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0178] All references cited in this specification are hereby incorporated by reference in their entirety. The following examples are solely for the purpose of illustrating one aspect of the inventionEXPERIMENTAL

[0179] The following examples are given for the purpose of illustrating various aspects of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of certain aspects, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.Example 1: Bispecific CD20-OR-CD19 CAR

[0180] A bispecific CAR was constructed to have, from N to C terminal, a signal sequence that directs CAR localization to the cell membrane, the CD20 scFv, a peptide linker, the CD 1968650W001 scFv, followed by a spacer (e.g., the TgG4 hinge domain), a transmembrane domain (e.g., the transmembrane domain of CD28), none or one or more co- stimulatory domains (e.g., the cytoplasmic domain of 4-1BB or CD28), and the cytoplasmic domain of CD3 , chain. To facilitate identification of CAR-expressing T cells by antibody staining, or elimination of CAR-expressing T cells, truncated epidermal growth factor receptor (EGFRt) can be linked to the CAR via a self-cleaving peptide (e.g., T2A).

[0181] The amino acid sequence of various components is as follows:68650W00168650W00168650W00168650W001

[0182] OR-gate CARs but not single-input CD19 CARs respond to Raji lymphoma cells that have undergone antigen escape, as shown in FIG. 2 by expression of CD69, CD 137 and CD1- 7a on the surface of the CAR-expressing T cells, and by the release of cytokines. The controls include a CD 19 Short single-input CD 19 CAR with IgG4 hinge as spacer; and CD20 Long single-input CD20 CAR with IgG4 hinge-CH2-CH3 as spacer. Various linkers were tested, including (G4S)1 (SEQ ID NO: 15), (G4S)4 (SEQ ID NO: 19), (SEQ ID NO:1, EAAAK)1, and (SEQ ID NO: 17, EAAAAK)3.

[0183] A comparison of cell lysis by single-input and OR-gate CAR-T cells after 4-hour coincubation with wildtype (WT; CD19+ / CD20+) or CD 19 — Raji (CD19- / CD20+) cells is shown in FIG. 3. The bispecificity was not compromised in OR-gate CARs. CAR-T cells were co-incubated with WT Raji or CD19+K562 targets for 24 hours, and the expression of relevant activation-induced antigens and release of cytokines are shown in FIG. 4 .

[0184] In vivo data, shown in FIG. 5, show OR-gate CARs abrogate the effects of antigen escape. In the survival of mice bearing WT or mixed Raji tumor xenografts and treated with T cells expressing no CAR or the single-input CD 19 CAR, the results showed that single-input CD 19 CAR was able to significantly extend the survival of animals engrafted with WT Raji tumors, and that OR-gate CARs are as efficient as single-input CD 19 CAR in targeting WT Raji lymphoma. However, only OR-gate CARs were able to significantly extend survival of animals bearing CD 19 — mutant tumors. OR-gate CARs are equally efficient against WT and CD 19 — mutant Raji tumors, thus rendering the T cells insensitive to antigen escape by target cells.

[0185] Shown in FIG 7, dual-targeting CD19 / CD20 CAR Tis an autologous, dual-targeting CD19 / CD20 CAR T-cell product candidate manufactured using enriched CD62L+cells to yield a final drug product comprising predominantly naive and central memory T cells.Example 2. A First Phase 1 Clinical Results of dual-targeting CD19 / CD20 CAR T Enriched in Naive and Central Memory T-Cells, for the Treatment of Large B-cell Lymphoma

[0186] Background: This example describes an ongoing study MPCT-012L (NCT05826535) which is a multi-cohort, open-label, dose-escalation (modified 3+3 design starting at 100M68650W001CAR+ cells) and expansion Phase 1-2 multi-center trial enrolling patients with LBCL, including DLBCL, HGBCL, tFL and PMBCL.

[0187] CD19-directed CAR T-cell therapies have revolutionized the treatment of B-cell lymphomas, though clinical experience shows many patients do not respond or relapse. More than 40% of patients never obtain a complete response (CR) to FDA-approved, CD19-targeted, CAR T-cell therapies (Neelapu et al., 2017; Abramson et al.. 2020). Approximately 50 to 60% of patients treated with CD19-targeted CAR T-cell therapies do not experience durable responses (Neelapu et al., 2023) and the median PFS rates in 3rdline LBCL remain only 6 to 7 months. Key reasons may include: Heterogeneous and potentially suboptimal antigen density (Maizner et al., 2020), CAR T-cell exhaustion which is associated with an effector-rich product (Westin et al. 2023) and Antigen escape due to single antigen targeting (Spiegel et al. 2021).

[0188] The Dual-targeting CD19 / CD20 CAR T cell was designed to maximize complete response (CR) rates, enhance cell persistence, reduce CAR T-cell exhaustion, and overcome variable CD19 antigen density. The autologous, dual-targeting CD19 / CD20 CAR T-cell product candidate is manufactured using enriched CD62L+cells to yield a final drug product comprising predominantly naive and central memory T cells (FIG. 7). The dual-targeting CAR described herein incorporates the same CAR construct as CART19 / 20, however, is manufactured using an improved and more efficient process. CART19 / 20 has reported durable responses in a single-institution Phase 1 trial (NCT04007029) (Larson et al., 2023; Puliafito et al. 2023).

[0189] Data from 13 patients with multiple histologies of B-cell non-Hodgkin lymphoma in the study of CAR 19 / 20 at UCLA was updated at the 2024 AACR Special Conference in Cancer Research with a 92% ORR and a 73% CR rate (Chen, 2024). The median PFS was 50.1 months, and the median overall survival was not reached with a median follow-up time of 32 months (range: 5.7 - NE).

[0190] A favorable safety profile was reported. Of note, two patients who achieved CR and relapsed (one after 18 months and one after 4 years of CR) were successfully retreated with CART 19 / 20 and achieved CR. Herein the initial data from a multi-center Phase 1-2 clinical trial evaluating the dual-targeting CD19 / CD20 CAR T is detailed.Methods68650W001

[0191] Cohorts include CAR T-naive patients in the 3rdline setting, CAR T-naive patients in the 2ndline setting, and CAR-T experienced patients. Data from the 3rdline CAR T-naive cohort are described here.

[0192] Eligibility for the 3rdline CAR T-naive cohort required 2 or more prior lines of therapy including exposure to an anti-CD20 monoclonal antibody and an anthracycline, and no prior treatment with an approved or investigational CAR T-cell product.

[0193] Patients received a standard lymphodepletion regimen (fludarabine 30 mg / m2and cyclophosphamide 500 mg / m2) and, after two rest days, a single flat dose (based on CAR+ cells) of dual-targeting CD19 / CD20 CAR T cells at the target dose level given in either the inpatient or outpatient setting. The tandem CD 19 / 20 CAR of the dual-targeting CD19 / CD20 CAR T was initially described by Zah and colleagues (Zah, 2016). A schematic is presented in FIG. 7A. The extracellular domain contains tandem scFv domains, one derived from the murine Leu 16 monoclonal antibody targeting CD20 and another derived from the murine FMC63 monoclonal antibody targeting CD 19, connected via a (Gly-Gly-Gly-GlySer)4 linker. This targeting region of the tandem bispecific molecule is attached through a short IgG4 hinge to a CD28-derived transmembrane region. The intracellular signaling region is comprised of the cytoplasmic co- stimulatory domain of human 4- IBB and the cytoplasmic domain of human CD3-^ at the C terminus. The transmembrane domain derived from human CD28 provides the physical link between the hinge and the intracellular domain.

[0194] The dual-targeting CD19 / CD20 CAR T cells are comprised of genetically modified autologous T cells reprogrammed to target cells that express CD 19 and / or CD20 antigens on the surface of normal B cells and B-cell tumors. The T cells are genetically modified through transduction with a lentiviral vector expressing bispecific scFv of murine anti-CD19 (FMC63) x CD20 (LEU16) antibodies and linked to CD28 transmembrane domain; the CD28 transmembrane domain is linked to an intracellular portion that contains T cell signaling (CD3- Q and co-stimulatory (4- IBB) domains. These intracellular domains play critical roles in different functions, including T cell activation, cytokine release, persistence in vivo, and antitumor activity. The dual-targeting CD19 / CD20 CAR T cells will be administered thawed after cryopreservation by intravenous (IV) injection.68650W001

[0195] The starting dose for this study is a single infusion of 1 x 108CD19 / 20 CAR-T cells based upon available data from 3 clinical studies of CD 19 / 20 bispecific CAR-T cells (Shah, 2019; Tong, 2020; Larson, 2023).

[0196] Unlike standard drugs that are metabolized, gene edited T cells are “living drugs” that can proliferate extensively in participants and, thus, the actual in vivo amount of CD 19 / 20 CAR T cells after engraftment and expansion will vary from participant to participant. Thus, the administered dose may underestimate the subsequent in vivo number dual-targeting CD19 / CD20 CAR T cells in a given participant.

[0197] The ability to manufacture the targeted dose for the majority of participants is an important consideration in the study. Taking into account the manufacturing feasibility, cellbased assay variability, and recoverable volume, a ± 20% variability in the dose volume is used for infusion.

[0198] Pre-infusion medications consisting of acetaminophen 650 mg orally, and diphenhydramine 25-50 mg orally or by IV, should be administered approximately 30 to 120 minutes prior to infusion. These medications may be repeated every 6 hours as needed.

[0199] Phase 1 will follow a modified 3+3 design with two planned dose levels (DLs): DL1 as 1 x 108(+ 20%) and DL2 as 3x 108(+ 20%) the dual-targeting CD19 / CD20 CAR T cells. If a dose level is safe based on the incidence of DLTs during a 28-day window. Participants in each dose level will be evaluated for DLTs occurring within the first 28 days following the completion of their respective dual-targeting CD19 / CD20 CAR T cells infusion. The analysis of a dose level will be based on the DLT evaluable participants. DLT evaluable participants are defined as receiving the target dose (± 20%) with a minimum of 28 days of follow-up. In Phase 1, the safety data is recorded to provide data for decisions on dose escalations / de- escalations and dose level prioritization. Phase 2 will initially consist of an expansion group of participants. Adverse reactions are considered Dose-Limiting Toxicity (DLTs) if they occur within the first 28 days following the infusion. Adverse events are graded according to ICANS All American Society for Transplantation and Cellular Therapy (ASTCT) Grade > 3 ICANS of any duration.68650W001Clinical Results

[0200] Twenty-three patients with LB CL were dosed with the dual-targeting CD19 / CD20 CAR Tin the CAR T-naive cohort with 2 or more prior lines of therapy and defined the Safety Analysis Set. Twenty-one patients were dosed with 100M (1 x 108) cells, and two at 300M (3x 108) cells.

[0201] The median age was 65 years (range, 21 - 88) and 16 (70%) were male. The majority of patients had a diagnosis of DLBCL 14 (61%). Most (57%, 13 / 23) reported advanced-stage disease at initial diagnosis and 48% (11 / 23) had elevated LDH at baseline.

[0202] Patients received a median of 2.8 lines of therapy (range, 2 - 6) and 53% (12 / 23) received bridging therapy. At the time of study entry, 39% (9 / 23) had an IPI score >3.

[0203] Table 2 Demographics & Baseline Characteristics of the CAR T-naive Cohort in the 3rd Line Setting68650W001

[0204] No cases of Grade 3 cytokine-release syndrome (CRS) were reported. Grade 1 and 2 CRS were reported in 70% (16 / 23) of patients and 50% (8 / 16) of those with CRS received tocilizumab.

[0205] Grade 3 ICANS was reported in 13% (3 / 23) of patients with a median time to complete ICANS resolution of 5 days, and rapid improvement to <Grade 3 promptly with standard therapy.

[0206] Four patients had Grade 3+ infections, all of which responded to treatment and resolved, and 17% (4 / 23) of patients had prolonged cytopenias.

[0207] Table 3: Key Safety Outcomes68650W001

[0208] Prolonged cytopenias were defined as Grade 3 or higher reported adverse events of neutropenia, anemia, thrombocytopenia or pancytopenia initiating or persisting on or after study day +28.

[0209] The efficacy evaluable population included 17 patients with LB CL, including DLBCL (n = 10), HGBCL (n = 4). and tFL (n = 3).

[0210] Patients were evaluable with a response assessment at Day 84 or later, or if they had a prior complete response or progressive disease.

[0211] The ORR is 94%, with a significant majority (71%) of patients achieving a CR by three months (Table 3, FIG. 8).

[0212] With a median follow-up of 6.3 months (range, 1.2 - 12.5), 71% of patients were in response at last follow-up. No deaths occurred during the study treatment period (prior to disease progression or subsequent anti-cancer therapy).

[0213] Table 4 Objective Response Rates68650W001

[0214] Two patients with T-cell histiocyte-rich large B-cell lymphoma histology were not included in the above analysis and had an ORR of 50%. FIG. 8: Individual patient trajectories demonstrate a 94% ORR and a 71% CR rate with 71% of patients in response at last followup with median follow up of 6.3 months of (range, 1.2 - 12.5 months). All patients were treated with the 100M cell dose except two patients who were dosed at the 300M cell dose.Patient and Product Cellular Kinetics

[0215] The dual-targeting CAR T cell product showed robust expansion in 16 patients evaluable for pharmacokinetic analysis. Peak cell expansion occurs between Days 7-28 post T-cell infusion (median Tmax= 10 days).

[0216] Median peak of expansion in 16 evaluable patients is 93,723 copies / pg gDNA (range 2,338 - 555,284). The dual-targeting CD19 / CD20 CAR T cells persist multiple weeks post infusion across multiple patients with median expansion at Day 28 of 11,766 copies / pg gDNA (range 55.7 - 555,284).

[0217] Median AUC (o-28 days) in 16 evaluable patients is 1,335,253 days x copies / pg gDNA (range 20,164 - 6,848,015).

[0218] FIG. 9 depicts time plots of individual patient PK concentration by droplet digital PCR (ddPCR) on DNA extracted from isolated peripheral blood mononuclear cells from 16 patients. Robust expansion with median peak of 93,723 copies / ug seen in treated CAR-T naive patients. Samples were analyzed in a duplex reaction at a DNA input of 50 ng per reaction using probes targeting FMC-63 and normalized using housekeeping gene SDC4. Intended timepoints of blood collection shown, actual timepoints vary by + / - 2 days. The dual-targ eting CAR T cells showed robust expansion in 16 patients evaluable for pharmacokinetic analysis. Peak cell expansion occurs between Days 7-28 post T-cell infusion (median Tmax = 10 days). Median6865OWDO1 peak of expansion in 16 evaluable patients is 93,723 copies / ug gDNA (range 2,338 - 555,284). CAR T cells persist multiple weeks post infusion across multiple patients with median expansion at D28 being 11,766 copies / ug gDNA (range 55.7 - 555.284). Median AUC <o-28 days) in 16 evaluable patients is 1,335,253 days x copies / ug gDNA (range 20,164 - 6,848,015).Dual-Targeting CD19 / CD20 CAR T Final Drug Product Characteristics

[0219] FIG. 10 depicts the final drug product from clinical lots (N = 23) is predominantly a CD3-rich product (median, 98%, range, 95 to 98%) comprising CD8 (median, 41%; range, 3 -71%) and CD4 (median, 57%; range, 23 - 96%) populations.

[0220] The CD3 component of the final drug product is a CD62L-rich product comprising naive and central memory populations (median, 91%; range, 82 - 99%).

[0221] The dual-targ eting CD19 / CD20 CAR T candidate has a manageable safety profile with no high-grade CRS and low rates of high-grade ICANS. Overall, adverse events were resolved with standard management algorithms.

[0222] FIG. 11. depicts a schematic of 3 + 3 dose escalation followed by dose expansion for the Phase 1- 2 Trial Design. Data evaluating the novel dual-targeting CD19 / CD20 CAR cell product candidate enriched for naive and central memory T cells, support the potential for a high rate of durable clinical outcomes with a favorable safety profile in CAR T-naive patients with LB CL in the 3rd-line setting. An objective response rate of 94% and a complete response rate of 71% were achieved after CAR T treatment in patients with CAR T-naive patients with LB CL who had received at least 2 prior lines of therapy.Example 3. Interim data from a Phase 1 trial of a CD19 / CD20 dual-targeting CAR T-cell candidate enriched for CD62L+stem-like cells achieves high rates of durable complete responses in R / R large B-cell lymphoma

[0223] Introduction: Durable complete response remains the therapeutic goal when treating patients with large B-cell lymphoma (LBCL) with CAR T-cell therapy. Despite the benefit of approved CAR T-cell therapies, approximately 50% of patients with relapsed / refractory (R / R) LBCL treated with CD19 CAR T-cell therapies progress by 6 months. In this Example, Phase68650W0011 data for an autologous dual-targeting CD19 / CD20 CAR T-cell candidate enriched for CD62L+naive and central memory T-cells is described. LYL314 (formerly IMPT-314) is designed to maximize response regardless of baseline antigen heterogeneity, while reducing CAR T-cell exhaustion and enhancing cell persistence. In this Example, data from patients treated in the ongoing third- and later-line setting (3L+) plus initial data from the second line (2L) setting from the Phase 1 / 2 study multi-center trial (NCT05826535) is presented.

[0224] Methods: Patients had R / R disease that was measurable prior to lymphodepletion and no prior CAR T-cell exposure. Patients treated in a 2L setting were required to have relapsed within 12 months of frontline therapy or had primary refractory disease. Following a standard lymphodepletion regimen of fludarabine and cyclophosphamide, patients received a single dose of LYL314. Investigator assessed responses were determined per Lugano Criteria.

[0225] Results: As of 28FEB2025, 45 CAR T-naive patients (31 3L+, 142L) with R / R LBCL received LYL314 (CD19 / CD20 dual-targeting CAR). Median age was 66 (21 to 87), 42% (19 / 45) had an International Prognostic Index of33, 67% (30 / 45) had ECOG status of 1, 53% (24 / 45) received bridging therapy, and 60% (27 / 45) had an elevated lactate dehydrogenase level at baseline. Median baseline tumor burden (sum of target lesion product diameters) was 13 cm2(2 to 180). Among treatment emergent adverse events, Grade 1 or 2 CRS was reported in 62% (28 / 45) patients, with no3Grade 3 CRS. Grade 1 or 2 ICANS was reported in 11% (5 / 45) patients and Grade33 ICANS was reported in 13% (6 / 45). All ICANS cases resolved promptly in a median of 5 days (2-12) with standard therapy. Grade 3 infections were reported in 13% (6 / 45) patients. There were no deaths on study related to LYL314. Thirty-one patients were response evaluable (Day 84 response assessment or died / progressed prior to Day 84). The overall response rate was 94% (29 / 31) including a complete response rate of 74% (23 / 31) with a median follow up of 7 months. Among those with a complete response, 76% (10 / 13) remained in complete response at the 6-month assessment. Robust CAR-T expansion was seen in PK evaluable patients (median AUCo-zsd of 1,317,204 days x copies / ug DNA).

[0226] Conclusions: A durable complete response rate of 76% was observed at the 6-month assessment in patients with LBCL treated with LYL314 in a Phase 1 / 2 multi-center trial. Additional results included a 94% overall response rate and a 74% complete response rate with a manageable safety profile. A pivotal trial of LYL314 is ongoing in patients treated in the 3L+ setting without prior CAR T-cell exposure (PiNACLE Trial).68650W001Example 4. Updated interim data from a Phase 1 trial of a CD19 / CD20 dual-targeting CAR T-cell candidate enriched for CD62L+ stem-like cells achieves high rates of durable complete responses in R / R large B-cell lymphomaThis Example describes updated Phase 1 clinical trial data from the clinical trial described in Example 3.

[0227] Results: As of 15 April 2025, 51 CAR T-naive patients (343L+, 172L) with R / R LBCL received LYL314 (CD19 / CD20 dual- targeting CAR). FIG. 12 shows a schematic of the study. Patient characteristics are shown in the Table 5 below.68650W001

[0228] As shown in Table 6 and FIG. 13, 3L+ CAR-Naive patients showed a high rate of durable complete responses with increasing duration of response with more follow-up time. Patients were evaluable for efficacy if they had a Day 84 or later response assessment, disease progression, or death from any cause. Two patients with T-cell histiocyte not included (1PR, 1 PD); histology no longer enrolled. Seven patients were dosed without Day 84 follow up, disease progression, or death.68650W001

[0229] 71% (10 / 14) of patients achieving a complete response remained in complete response at > 6 months.

[0230] Early data from high-risk 2L patients demonstrate high overall response rate. See e.g., Table 7. In particular, 100% (7 / 7) of patients with CR remain in CR at last assessment, including 3 / 3 at > 6 months. Additionally, in patients with primary refractory disease, a difficult to treat population, 70% (7 / 10) achieved a complete response.68650W001

[0231] The results showed a manageable safety profile allowing for outpatient administration. Table 8 summarizes the adverse events. 69% of patients with CRS received tocilizumab. ICANS rate has decreased since the introduction of prophylactic dexamethasone (N = 15; data not shown). No deaths were related to LYL314.6865OWDO1

[0232] LYL314 showed robust expansion (N=51) with peak CAR T cells of 70,685 copies / / zg (1,387 - 569,039), AUC CAR T cells of 819,198 days x copies / / rg (14,596 - 9,109,115), and time to peak of 10 days (7 - 28) (FIG. 15 A).

[0233] Final drag product showed a median of 95% CD62L+cells comprising naive and central memory populations (median, 95%; range, 84.1% - 99.4%) (FIG. 15C).

[0234] As shown in FIG. 15B, patients had rapid and durable B-cell aplasia through Month 6 and up to the Month 12 assessment for patients with available data using a highly-sensitive and robust method.

[0235] Three selected patient cases are summarized below for illustration. Investigator assessed responses are reported.

[0236] 1. High-risk patient with rapid and durable complete response. A 64-year-old male withDLBCL transformed from follicular lymphoma with a palpable neck mass which demonstrated rapid reduction by Day 5 and a CR at Day 28 that remains ongoing at 12+ months. Patient history: Prior Therapy : Relapsed after second-line salvage chemotherapy and autologous6865OWDO1 stem cell transplant. Medical History: Prior myocardial infarction with stent. Baseline Labs: LDH>ULN; Adverse Events: CRS Grade 1 (resolved without tocilizumab or dexamethasone); COVID-19 infection resolved as outpatient.

[0237] 2. Low CD19-antigen DLBCL with complete response. 52-year-old female withDLBCL that relapsed 3 months after first-line therapy. Lymphoma Characteristics: CD19 low, and CD20 positive DLBCL by immunofluorescence. Day 28 complete response that is ongoing at 3+ months.

[0238] 3. 85-year-old female, primary refractory disease, double-hit HGBCL. Patient and lymphoma characteristics: Prior Therapy: R-CHOP x 6 cycles; Primary refractory disease Deauville 5) confirmed on PET / CT. Lymphoma Characteristics: Double hit (translocations of MYC; and BCL2) lymphoma with multiple sites of extranodal disease (pleura and soft tissue). Baseline Labs: LDH>ULN. Adverse Events: Grade 1-2 CRS treated with tocilizumab, Grade 1 ICANS with full recovery in less than 24 hours. Patient showed complete response at Day 28 with CR ongoing at 6+ months.

[0239] Conclusions: Results from a Phase 1 / 2 multi-center study evaluating LYL314 in 3L+ and 2L CAR-naive patients show that LYL314 is a promising CAR T-cell candidate for large B-cell Lymphoma. High response rates were observed in high-risk CAR-naive patients with ORR of 88% and a CR rate of 72% in 3L+ setting. 71% of patients with complete response remained in complete response at > 6 months in the 3L+ setting. There was a manageable safety profile appropriate for outpatient administration with no Grade 3 CRS and low rates of Grade 3 ICANS. Robust CAR T-cell expansion was seen with the final drug product enriched for stem-like cells (CD62L+) and a rapid and durable depletion of B cells was observed.Example 5. Rondecabtagene Autoleucel, an Autologous, Dual-Targeting CD19 / CD20 CAR T-Cell Candidate Manufactured from CD62L+ Enriched T Cells, Achieves Durable Responses in Patients with Large B-Cell Lymphoma

[0240] Background: Single-antigen CD19-targeting chimeric antigen receptor (CAR) T-cell therapies have become a standard of care for patients with large B-cell lymphoma (LB CL) that68650W001 relapses or is refractory to chemoimmunotherapy, with a goal of durable complete response. Rondecabtagene autoleucel (ronde-cel, LYL314) is an autologous, dual- targeting CD19 / CD20 CAR T-cell product candidate manufactured from CD62L+ enriched naive and central memory T cells. Ronde-cel is designed to maximize response regardless of baseline antigen heterogeneity, while reducing CAR T-cell exhaustion, enhancing T-cell persistence, and mitigating antigen loss. In this Example, updated data is presented from the ongoing Phase 1 / 2 multi-center trial (NCT05826535) as described in Examples 3 and 4, with a focus on patients with high-risk, second- line (2L) LB CL, as well as extended follow up of patients treated in the third- or later-line (3L+) setting. Enrollment is ongoing and additional data will be available at the time of the conference.

[0241] Methods: Patients had relapsed or refractory (R / R) disease that was measurable prior to lymphodepletion (LD). Patients treated in a 2L setting were required to have relapsed within 12 months of frontline therapy or had primary refractory disease. Patients with 3L+ LB CL are now being enrolled into a pivotal study (PiNACLE). No baseline CD 19 or CD20 antigen testing was required for enrollment and there was no upper age limit. Eollowing a standard 3- day LD regimen of fludarabine and cyclophosphamide, patients received a single dose of ronde-cel, in the outpatient or inpatient setting, at the recommended Phase 2 dose (RP2D) of 100 x 106CAR cells. Investigator assessed responses were determined per Lugano Criteria.

[0242] Results: As of June 27, 2025, 60 patients had received ronde-cel (23 2L; 37 3L+). Median age was 65 years (range 21-87), 65% (39 / 60) had ECOG status of 1, 40% (24 / 60) had an International Prognostic Index of33 at time of enrollment, 55% (33 / 60) had Stage IV disease, 42% (25 / 60) had elevated lactate dehydrogenase (LDH) at baseline, and 55% (33 / 60) received bridging therapy. Median (IQR) baseline tumor burden (sum of target lesion product diameters) was 18.2 cm2(6 - 42). In the 2L setting, 70% (16 / 23) of patients had primary refractory disease. Lorty-six patients were response evaluable: 15 and 31 in the 2L and 3L+ settings, with median follow up of 7 and 10 months, respectively. Among patients in 2L, the overall response rate was 87% (13 / 15) including a complete response rate of 60% (9 / 15), with 100% (7 / 7) of patients with complete response remaining in complete response at > 6 months. Among the patients in 3L+, the overall response rate was 90% (28 / 31) and the complete68650W001 response rate was 71 % (22 / 31). Grade 1 or 2 cytokine release syndrome (CRS) was reported in 62% (37 / 60) patients, with no3Grade 3 CRS, and 42% (25 / 60) of all patients receiving tocilizumab. Grade 1 or 2 immune effector cell-associated neurotoxicity syndrome (ICANS) was reported in 12% (7 / 60) of patients. Grade33 ICANS was reported in 13% (8 / 60) of patients, including 7% (1 / 15) of patients after the introduction of routine dexamethasone prophylaxis, and resolved promptly with standard therapy (median time to < Grade 3 was 2.5 days). Grade 3 infections were reported in 13% (8 / 60) patients. There were no deaths on study related to ronde-cel.

[0243] Conclusions: Ronde-cel achieved high overall response and complete response rates with an encouraging safety profile in high-risk patients with LB CL in a Phase 1 / 2 multi-center trial. Complete responses among patients in 2L were durable at 6 months (100%). A singlearm pivotal trial of ronde-cel is ongoing in patients treated in the 3L+ setting (PiNACLE), and, based on these results, a Phase 3 head-to-head, randomized controlled trial of ronde-cel versus investigator’s choice of approved CD 19 CAR T-cell therapy (lisocabtagene maraleucel or axicabtagene ciloautoleucel) has been initiated (PiNACLE-H2H).

[0244] Updated patient characteristics and safety data as of September 5, 2025 are outlined in Tables 9 and 10 below.68650W00168650W00168650W001Data will continue to be updated as the clinical trials evaluating ronde-cel progress.Example 6. CD62L Enrichment Achieves Robust Expansion and Memory Phenotype PostInfusion in Patients with LBCL Treated with Rondecabtagene Autoleucel, an Autologous, Dual-Targeting CD19 / CD20 CAR T-Cell Candidate

[0245] Background: Durable complete response remains the therapeutic goal when treating patients with large B-cell lymphoma (LBCL) with chimeric antigen receptor (CAR) T-cell therapy. Despite the benefit of approved CD19 CAR T-cell therapies, approximately 50% of patients with third- or later- line (3L+) relapsed or refractory (R / R) LBCL treated with CD 19 CAR T-cell therapies progress by 6 months. Higher memory marker expression and lower exhaustion marker expression in FDA- approved CD 19 CAR T-cell drug products have been associated with favorable clinical outcomes (Deng et al Nature Medicine, 2020, Vol 26 (12), 1878-1887). CD62L is a known cell surface marker for stem-like memory and central memory T-cells (Gattinoni, L., Speiser, D., Lichterfeld, M. et al. Nat Med 23, 18-27 (2017)). Rondecabtagene autoleucel (ronde-cel, LYL314) is an autologous, dual-targeting CD19 / CD20 CAR T-cell therapy product candidate manufactured from CD62L+ enriched cells. This manufacturing process is designed to maximize memory properties, reduce T-cell exhaustion, and enhance cell persistence in the drug product, and does not extend conventional manufacturing time. An ongoing Phase 1 / 2 multi-center trial (NCT05826535) (see Examples 3-5) in 2L and 3L+ LBCL is evaluating ronde-cel. which has achieved high rates of durable responses (Merchant et al 2025 ICML abstract). Here, translational data is presented in patients treated in the 2L and 3L+ setting, highlighting that CD62L+ enrichment achieves greater memory phenotype expression and in vivo cell expansion compared to published data from68650W001FDA approved CAR T-cell therapies in LBCL. Enrollment is ongoing and additional data will be available at the time of the conference.

[0246] Methods: Phenotype of ronde-cel drug products was assessed by flow cytometry and single-cell RNAseq. Peripheral blood (PB) was collected post infusion at multiple time-points, and CAR T-cell pharmacokinetics (PK) (ddPCR) and phenotype (flow cytometry) were assessed.

[0247] Results: Ronde-cel products (N = 53) are composed of, on average, 99% CD3+ cells (range: 89 - 100%), 59% CAR+ cells (17 - 79%), and 95% CD62L+ of CD3+ cells (84 - 99%). Transcriptomic analyses on the CD8+CAR+ compartment of ronde-cel products (N = 19) shows higher expression of memory-related genes such as CD62L, IL7R, and CCR7 and lower expression of exhaustion-associated genes such as TIG IT, PD-1, and LAG3 compared to an approved CD19 CAR-T therapy (Li et al 2023 Cancer Cell, 41(11): 1835- 1837). Beyond individual genes, gene set analyses (Hanzelmann, S., Castelo, R. & Guinney, I. GSVA: gene set variation analysis for microarray and RNA-Seq data. BMC Bioinformatics 14, 7 (2013).) reveal that stem-like gene sets (Jansen et al Nature 2019, 576(7787):465-470) and effectormemory gene sets (Sade-Feldman et al, Cell, 2018 Nov l;175(4):998-1013.e20) are significantly upregulated (adjusted p values = 6.4e-6 and 2.5e-3, respectively) in ronde-cel products compared to the approved CD19 CAR-T cell therapies. Conversely, exhaustion- associated gene sets are downregulated (adjusted p value = 1.8e-4) in ronde-cel products. Flow cytometry profiling on PB mononuclear cells from Day 14 after ronde-cel infusion (N = 14) showed that CD8+CAR+ cells are predominantly of memory phenotype (median of 98% and standard deviation of 19%, as defined by CD45RO+ / CD45RA-).

[0248] The enhanced memory phenotype of ronde-cel products enabled robust CAR T-cell expansion post infusion (N = 54) with a peak (Cmax) of 83,423 copies / pg (1,387 - 569,039), AUC of 898,191 days x copies / pg (14,596 - 9,109,115), and time to peak of 10 days (7 - 28). Median peak expansion (Cmax) and exposure (AUC) for ronde-cel is at least 3x higher than FDA approved CAR T-cell therapies (Abramson et al 2020, Lancet 396(10254):839-852; Schuster et al N Engl J Med 2019;380:45-56). Finally, higher CAR T-cell exposure (AUC)68650W001 was positively correlated with increased effector-memory gene set scores measured in ronde- cel products (r = 0.65, p = 0.01, N = 15).

[0249] Conclusions: Ronde-cel manufactured after CD62L enrichment resulted in increased stem-like and effector-memory properties in the infusion product compared to FDA approved CD 19 CAR T-cell therapies in LBCL. These properties are positively correlated with robust expansion observed in patients and a high memory phenotype post infusion. These findings support the potential of ronde-cel to achieve improved clinical outcomes through enhanced product characteristics. A single-arm pivotal trial of ronde-cel is ongoing in patients treated in the 3L+ setting (PiNACLE), and a Phase 3 head-to-head, randomized controlled trial of ronde- cel versus investigator’s choice of approved CD 19 CAR T-cell therapy (lisocabtagene maraleucel or axicabtagene ciloautoleucel) has been initiated (PiNACLE-H2H).Example 7. Updated Analysis Further Confirms CD62L Enrichment Achieves Robust Expansion and Memory Phenotype Post- Infusion in Patients with LBCL Treated with Rondecabtagene Autoleucel, an Autologous, Dual-Targeting CD19 / CD20 CAR T-Cell Candidate

[0250] In this Example, updated translational data is presented from the ongoing Phase 1 / 2 multi-center trial (NCT05826535).

[0251] To assess the phenotype of ronde-cel in comparison to FDA approved CAR-T products,Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) was performed on 34 ronde-cel products after sorting for CD8+CAR+ cells.

[0252] Compared to reference cells from four public studies (i.e. described in Deng, Q. et al (2020). Nature Medicine, 26(12), 1878-1887, Haradhvala, N. J. et al (2022). Nature Medicine, 28(9), 1848-1859. https: / / doi.org / 10.1038 / s41591-022-01959-0, Li, X. et al. (2023). Cancer Cell, 41(11), 1835-1837. https: / / doi.Org / 10.1016 / j.ccell.2023.08.015, and Yu, X. et al. (2025). Journal for ImmunoTherapy of Cancer, 13, e011807. https: / / doi.org / 10.1136 / jitc-2025-011807.), the CD8+CAR+ cells of ronde-cel (N=34) had higher expression of memory markers (FIG. 16A, 16B, 16C) and expression of a memory68650W001 geneset (FTG. 16D) and lower expression of short-lived effector markers (FIG. 17A, 17B, 17C) and an effector geneset (FIG. 17D).

[0253] To assess the peripheral pharmacokinetics (PK) of ronde-cel in the participants, peripheral blood was collected at various timepoints post ronde-cel infusion. Initial blood samples were collected at day -5 (prior to lymphodepletion) before ronde-cel infusion and then on Days 4, 7, 10, 14, 21, 28, 56, 84, 180, and every 6 months up to 2 years after infusion. Genomic DNA was isolated from spun whole blood and ronde-cel CAR copies were quantified using a digital-droplet (ddPCR) assay. Ronde-cel CAR vector copies per ug gDNA was calculated using SDC4 as an internal reference gene to calculate total gDNA per sample.

[0254] This analysis confirmed that ronde-cel CAR T cell expansion was observed in all participants evaluated. As shown in FIG. 18, the median peak value for the 67 participants analyzed was 85,888 copies / ug DNA, and peak expansion was seen on average 14 days post ronde-cell infusion.

[0255] To assess the association between product phenotype and PK of ronde-cel, a linear model was fit between the peak expansion and the pseudobulk expression of IL7R (FIG. 19A) and a memory associated geneset (FIG. 19B). Both linear models were significant (p< .05), confirming that higher expression of memory associated genes correlates with greater expansion of ronde-cel in the patient post-infusion.

[0256] To assess the phenotype of ronde-cel post-infusion in comparison to FDA approved CAR-T products, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE- Seq) was performed on 11 PBMC samples collected four weeks post-infusion of ronde-cel. The PBMCs were sorted for CD8+CAR+ cells for library preparation.

[0257] As a comparator, CITE-seq from four patients treated with axi-cel from Cao, G. et al (2025). bioRxiv. https: / / doi.org / 10.1101 / 2025.03.05.641715 was used. For both the ronde-cel and axi-cel data, samples with fewer than 500 CD8+CAR+ cells were excluded due to low signal to noise at that sample size.

[0258] A memory phenotype was identified in the clustering of each dataset by low expression of GZMB and KLRG1. The frequency of that phenotype is higher in ronde-cel than axi-cel, showing that ronde-cel retains a higher proportion of memory cells four weeks after infusion (FIG. 20A). Pseudobulk comparison of cytokine expression shows that ronde-cel has higher expression of the pro-inflammatory cytokines IFNy and TNF (FIG. 20B and FIG. 20C).68650W001

[0259] Ev vivo assays on PBMCs collected from patients 56 days post-infusion of ronde-cel were performed to demonstrate the durable functionality of ronde-cel. PBMCs collected from three patients 56 days after infusion were co-cultured up to 5 days with a Nalm6 tumor cell line. The cells were cultured at a ratio of one CAR-T cell to one Nalm6 cell.

[0260] To assess the proliferative capacity of the ronde-cel, the PBMCs were stained with a Cell Trace Violet (CTV) dye at the beginning of the co-culture and then flow cytometry was conducted on day 5 of the co-culture to measure the percentage of CAR+ and CAR- cells that proliferated over the course of the co-culture. Greater than 80% of the CAR+ cells from all 3 patients proliferated in the co-culture while <10% of the CAR- cells proliferated (FIG. 21A). This demonstrates that the ronde-cel CAR+ cells from 56 days post-infusion have the capacity to robustly proliferate upon stimulation with lymphoma tumor cells.

[0261] An ex vivo tumor killing assay was used to measure the killing capability of ronde-cel CAR-T cells 56 days post-infusion. Thawed PBMCs were co-cultured with Nalm6 tumor cell line for three days in an incucyte at a ratio of 1:1 to measure the number of tumor cells killed. As depicted in FIG. 2 IB the PBMCs from 3 patients killed all tumor cells in 3 days, while the Nalm6 cell line alone expanded 4x in the same timeframe. IFNg secretion by the CAR+ cells from the thawed PBMCs was demonstrated using Meso Scale Diagnostics (MSD) at day 3 of the co-culture (see FIG. 21C). Combined, these ex-vivo results demonstrate proliferation, killing and cytokine secretion of ronde-cel from 56 days post-infusion to patients.

Claims

68650W001CLAIMSWhat is claimed is:

1. A method of treating a cancer in a subject in need thereof comprising administering to the subject an effective amount of a population of cells expressing a polypeptide comprising a CD19 / CD20 chimeric antigen receptor (CAR), wherein the CAR comprises from N- to C- terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from a sequence of SEQ ID NO: 3; b) a (G4S)4linker (SEQ ID NO: 19) c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co-stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein between about 60% and about 99% of the population of cells expressing the polypeptide are CD62L+.

2. The method of claim 1, wherein at least about 65% of the population of cells expressing the polypeptide are CD62L+.

3. The method of claim 1, wherein after administration of the population of cells, the subject has a response wherein the response comprises a complete response, a partial response, or stable disease.

4. The method of claim 3, wherein a duration of the response after administration of the population of cells is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or more.

5. The method of claim 1, wherein prior to administration of the population of cells, the subject has received a lymphodepleting chemotherapy.

6. The method of claim 5, wherein the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, bendamustine, or any combination thereof.68650W0017. The method of claim 6, wherein the lymphodepleting chemotherapy comprises cyclophosphamide at about 500 mg / m2 and fludarabine at about 30 mg / m2 for three days.

8. The method of claim 6, wherein the lymphodepleting chemotherapy comprises cyclophosphamide at about 300 mg / m2 and fludarabine at about 30 mg / m2 for three days9. The method of any one of the preceding claims, wherein the effective amount is a therapeutically effective or pharmaceutically effective amount.

10. The method of any one of the preceding claims, wherein the effective amount of cells is a dose of 1 x 108(± 20%), 2 x 108(± 20%), or 3x 108(± 20%) cells.

11. The method of claim 1, wherein (a) the cancer is large cell B cell lymphoma; (b) the subject has R / R disease that was measurable prior to a lymphodepletion; and (c) the subject has no prior CAR T-cell exposure.

12. The method of claim 1, wherein the subject (a) has had no more than frontline therapy;(b) relapsed within 12 months of the frontline therapy; or (c) has had primary refractory disease.

13. A method of treating DLBCL, PMBCL, HGBL, Grade 3bFL, of tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6; f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 60% of the population of cells expressing the polypeptide are CD62L+.

14. The method of claim 13, wherein at least about 80% of the population of cells expressing the polypeptide are CD62L+.

15. The method of claim 13, wherein the bispecific CD19 / CD20 CAR T cells are produced in a closed system method of manufacturing comprising the steps of: a) isolating CD62L+ cells68650W001 from a starting population of cells, thereby obtaining a population of naive / memory T (TN / MEM) cells, wherein a depletion of CD14+ / CD25+ cells is not performed; b) contacting said population of TN / MEM cells with a transactivating agent to obtain a population of activated TN / MEM cells; c) transducing said population of activated TN / MEM cells with a viral construct to obtain a population of transduced cells, wherein transducing is performed in the absence of at least one transduction enhancer selected from the group consisting of: polybrene, protamine sulfate, LentiBoost™, Vectofusin-1, and poloxamer; d) expanding said population of transduced cells; and e) optionally removing the transactivating agent.

16. The method of claim 15, wherein the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide; or the viral construct does not include a polynucleotide encoding a truncated EGFR protein; or the viral construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) polynucleotide and the viral construct does not include a polynucleotide encoding a truncated EGFR protein.

17. The method of claim 13, wherein the subject has previously received one prior line of therapy.

18. The method of claim 13, wherein the subject has previously received two prior lines of therapy.

19. The method of claim 13, wherein the subject achieves a complete response for at least 6 months following administration of the T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR.

20. The method of claim 13, wherein the subject achieves a complete response for at least 8 months following administration of the T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR.

21. The method of claim 13, wherein the subject is CAR-experienced and has received at least 2 or at least 3 prior lines of therapy.

22. The method of any one of claims 13-21, wherein the effective amount of cells is a dose of 1 x 108(± 20%), 2 x 108(± 20%), or 3x 108(± 20%) cells.

23. The method of claim 13, wherein (a) the cancer is large cell B cell lymphoma; (b) the subject has R / R disease that was measurable prior to a lymphodepletion; and (c) the subject has no prior CAR T-cell exposure.

24. The method of claim 15, wherein steps a-d take no more than between 8 and 10 days.6865OWDO125. The method of claim 15, wherein the T cells expressing the polypeptide are administered to the subject no more than between 16 and 18 days from the day source T cells are collected from the subject.

26. A method of treating DLBCL, PMBCL, HGBL, Grade 3bFL, of tFL in a subject in need thereof, comprising administering 100 x 106(± 20%), 200 x 106(± 20%) or 300 x 106(± 20%) T cells expressing a polypeptide comprising a bispecific CD 19 / 20 CAR wherein the CAR comprises from N- to C-terminus: a) an anti-CD20 scFv comprising a variable light domain and a variable heavy domain from SEQ ID NO: 3; b) a (G4S)4 linker or a (G4S)I linker; c) an anti-CD 19 scFv comprising a variable heavy domain and a variable light domain from a sequence of SEQ ID NO: 4; d) a spacer of SEQ ID NO: 5; e) a transmembrane domain of SEQ ID NO: 6: f) a co- stimulatory domain of SEQ ID NO: 8; and g) a CD3-zeta cytoplasmic signaling domain of SEQ ID NO: 9, wherein at least about 60% of the population of cells expressing the polypeptide are CD62L+ and wherein the T cells expressing the polypeptide are administered to the subject no more than between 16 and 18 days from the day source T cells are collected from the subject.

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