Methods of treating multiple sclerosis with CD19-CD20 dual car-t cells

Abstract

The present disclosure provides methods of treating multiple sclerosis (e.g. progressive MS or relapsing remitting MS) by administering a chimeric antigen receptor T cell (CAR-T cell) comprising an anti-CD20 chimeric antigen receptor and an anti-CD19 chimeric antigen receptor. The present disclosure also provides a dose infusion bag comprising a composition comprising CAR-T cells comprising an anti-CD20 chimeric antigen receptor and an anti-CD19 chimeric antigen receptor.

Description

Attorney Docket No.: 000218-0094-W01METHODS OF TREATING MULTIPLE SCLEROSIS WITH CD19-CD20 DUAL CAR-T CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 720,075, filed November 13, 2024, the contents of which are herein incorporated by reference in their entireties.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on November 12, 2025, is named “000218-0094-W01-SL.xml” and is 10,976 bytes in size.TECHNICAL FIELD

[0003] The present disclosure pertains to the field of treatment of multiple sclerosis (MS) (e.g. progressive MS (PMS) or relapsing MS (RMS)) using dual chimeric antigen receptor T (CAR-T) cells comprising a chimeric antigen receptor (CAR) encoding an anti-CD20 antibody and a CAR encoding an anti-CD19 antibody.BACKGROUND

[0004] Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by chronic inflammation, demyelination, and progressive neurologic disability (Lassmann and Bradl 2017). MS affects approximately 2.8 million people globally, typically presenting in early adulthood with a mean age of onset between 20-30 years old and progressing throughout a patient’s life (Walton et al. 2020; McGinley et al. 2021). Common symptoms of MS include fatigue, motor and coordination impairments, sensory disturbances, visual problems, and cognitive changes. MS can manifest in several subtypes, including relapsing-remitting MS (RRMS), which is defined by episodes of new or increasing symptoms followed by periods of remission; primary progressive MS (PPMS), marked by gradual, steady progression of symptoms without relapses; and secondary progressive MS (SPMS), which initially presents as RRMS before transitioning into a progressive phase (Haki etAttorney Docket No.: 000218-0094-W01al. 2024). SPMS can be active (aSPMS), with relapses and / or new magnetic resonance imaging (MRI) activity, or non-active (naSPMS), with no relapses or new MRI activity. Relapse pathology is dominated by focal demyelinating inflammatory attacks while disease progression, which can occur in all MS subtypes, is marked by an increase in neurodegeneration and neurologic disability (Mahad et al. 2015).

[0005] There are over 20 disease-modifying therapies (DMTs) approved to treat MS, including immunomodulators (e.g., interferons, glatiramer acetate, dimethyl fumarate, teriflunomide), cell migration modulators (e.g., fmgolimod, siponimod, natalizumab), and cell-depleting therapies (e.g., alemtuzumab, cladribine, ocrelizumab, ofatumumab) (Piehl 2021). Additionally, corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone) are often administered during relapses to reduce inflammation (Smets et al. 2017). High-efficacy therapies including ofatumumab, ocrelizumab, natalizumab, alemtuzumab, and cladribine have been successful in substantially reducing relapsing pathology but have limited ability to cross the blood brain barrier (BBB), and a significant proportion of patients taking these drugs experience worsening disability. Further, these therapies require chronic immunosuppression, which carries long-term risks including a higher rate of serious infections and hyperglobulinemia (Sacco and Abraham 2018; Peters and Longbrake 2022). Therefore, a high unmet need remains for safer and more effective treatments to improve patient outcomes.SUMMARY

[0006] In a first aspect, the present disclosure provides methods of treating multiple sclerosis (MS) in a human subject in need thereof comprising administering a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) to the subject.

[0007] In a second aspect, the present disclosure provides the use of a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) in the manufacture of a medicament for treating multiple sclerosis (MS) in a human subject in need thereof.

[0008] In a third aspect, the present disclosure provides a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) for use in treating multiple sclerosis (MS) in a human subject in need thereof.Attorney Docket No.: 000218-0094-W01

[0009] In some embodiments of any of the above aspects, the MS is progressive MS (PMS). In some embodiments, the PMS is primary PMS (PPMS) or non-active secondary PMS (naSPMS). In some embodiments, the PMS is PPMS. In some embodiments, the PMS is naSPMS. In some embodiments, the MS is relapsing MS (RMS). In some embodiments, the RMS is relapsing remitting MS (RRMS) or active secondary PMS (aSPMS). In some embodiments, the RMS is RRMS. In some embodiments, the RMS is aSPMS. In some embodiments, the MS is diagnosed according to the revised 2017 McDonald Criteria.

[0010] In some embodiments of any of the above aspects, the CAR-T cell is administered to the subject intravenously or is formulated to be administered to the subject intravenously. In some embodiments of any of the above aspects, the CAR-T cell is administered as a single dose or is formulated to be administered as a single dose. In some embodiments, the CAR-T cell is administered in multiple doses or is formulated to be administered in multiple doses. In some embodiments, each of the multiple doses is administered between 3 and 5 years after the previous dose. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106cells and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106cells and about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 5 x 106cells and about 1.2 x 109cells.

[0011] In some embodiments of any of the above aspects, administration of the CAR-T cell results in (a) a detectable number of CAR transgene copies in the blood of the subject, (b) a detectable number CAR-T cells in the blood of the subject, (c) the depletion of B-cells in the blood or cerebrospinal fluid (CSF) of the subject by more than 90 percent as compared to prior to treatment, (d) a reduction in the number of gadolinium lesion counts and no new or enlarging T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment, (e) no disability progression measured by the composite disability progression score confirmed at 12 or 24 weeks (24w-cCDP or 12w-cCDP) after treatment, or (f) an improvement in MS-associated biomarkers (e.g., oligoclonal bands, NfL, and GFAP) as compared to prior to treatment.Attorney Docket No.: 000218-0094-W01

[0012] In some embodiments of any of the above aspects, administration of the CAR-T cell results in (a) an incidence of Grade 2 cytokine release syndrome (CRS) less than 30%, (b) an incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) less than 20%, (c) an incidence of hemophagocytic lymphohistiocytosis (HLH) less than 10%, (d) a risk of serious infection no more than 10% more compared to the incidence of infection resulting from standard of care 6 months after administration of the CAR-T cell, (e) an incidence of immune rejection and graft-versus-host disease (GVHD) less than 10%, (f) an incidence of progressive multifocal leukoencephalopathy (PML) less than 10%, (g) an incidence of CAR T cell-driven worsening of MS in less than 20% of subjects administered the CAR-T cell, or (h) an incidence of new T-cell malignancies less than 0.1%.

[0013] In some embodiments of any of the above aspects, the subject has not been previously administered a therapeutic agent to treat MS. In some embodiments of any of the above aspects, the subject has been previously administered one or more therapeutic agents to treat MS. In some embodiments, the subject is refractory to the one or more previously administered therapeutic agents. In some embodiments, the one or more previously administered therapeutic agents is selected from an immunomodulator, a cell migration modulator, a lymphocyte depleting therapy, a kinase inhibitor, or a corticosteroid. In some embodiments, the immunomodulator is selected from the group consisting of interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, teriflunomide, and Mitoxantrone. In some embodiments, the cell migration modulator is selected from the group consisting of fmgolimod, siponimod, ozanimod, and natalizumab. In some embodiments, the lymphocyte depleting therapy is selected from the group consisting of alemtuzumab, cladribine, ocrelizumab, rituximab, ublituximab, and ofatumumab. In some embodiments, the kinase inhibitor is selected from the group consisting of baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, and fenebrutinib. In some embodiments, the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.

[0014] In some embodiments of any of the above aspects, the CAR-T cell is administered or formulated to be administered in combination with one or more additional therapeutic agents. In some embodiments, the CAR-T cell is administered or formulated to be administered sequentially with the one or more additional therapeutic agents. In some embodiments, the one or more additional therapeuticAttorney Docket No.: 000218-0094-W01agents is selected from an immunosuppressant, a corticosteroid, a kinase inhibitor, a cytokine blocker, or a combination thereof. In some embodiments, the immunosuppressant is selected from the group consisting of cyclophosphamide, tocilizumab, siltuximab and anakinra. In some embodiments, the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone. In some embodiments, the kinase inhibitor is ruxolitinib. In some embodiments, the cytokine blocker is selected from the group consisting of ruxolitinib, etoposide, or emapalumab.

[0015] In some embodiments of any of the above aspects, the subject receives a lymphodepleting chemotherapy (LDC) regimen prior to the administration of CAR-T cell. In some embodiments, the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) and fludarabine (0-30 mg / m2IV) for 3 consecutive days. In some embodiments, the LDC regimen is administered on Days -5, -4, and -3, and the CAR-T cells is administered or to be administered on Day 1.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 provides a diagram showing the anti-CD19 and anti-CD20 CAR components of the P-CD19CD20-ALLO1 Transposon. [CAR = chimeric antigen receptor; cHS4 = chicken hypersensitive site 4; DHFR = dihydrofolate reductase; EEF1A1 = Eukaryotic translation elongation factor 1 alpha 1; HBB = hemoglobin subunit beta; iCasp9 = inducible caspase 9; ITR = inverted terminal repeat; nt = nucleotides; SV40 poly(A) = simian virus 40 polyadenylation; T2A = Thosea asigna virus 2 A, UTR = untranslated region; VH = variable heavy chain],

[0017] Figure 2 provides an illustration of P-CD19CD20-ALLO1 chimeric antigen receptor (CAR) on the surface of the T cell membrane.

[0018] Figures 3A and 3B provide graphs showing the % lysis of antigenpositive (Raji) tumor cells (Figure 3 A) and antigen-negative (K562) tumor cells (Figure 3B) by P-CD19CD20-ALLO1 across Effector to Target (E:T) ratios of 20:1-0.039:1. Each data point shows average of triplicates for Raji cell killing and average of duplicates for K562 cell killing.

[0019] Figures 4A-4D provide graphs showing IFN-y (Figures 4A and 4B) and Granzyme B (Figures 4C and 4D) levels in supernatants from coculture assays ofAttorney Docket No.: 000218-0094-W01P-CD19CD20-ALLO1 CAR-T cells (“Lot X (CAR-T)”) and mock transfected controls (“Lot X (mock)”) with either antigen-positive cells (Raji) (Figures 4A and 4C) or antigen-negative cells (Jurkat) (Figures 4B and 4D).

[0020] Figure 5 provides the results of a flow cytometry-based cytotoxicity assay against PBMC from MS patients. Cytotoxic activity of 3 lots of P-CD19CD20-ALLO1 CAR-T cells against peripheral blood mononuclear cell (PBMC) samples isolated from MS patients at E:T ratios of 1:1 and 5:1 was evaluated by flow cytometry after 48 hours co-culture. Each cell population in the PBMC sample was quantified using Countbright™ beads and numbers were normalized to the PBMC samples alone. Abbreviations: CAR-T = chimeric antigen receptor T cell; E:T = Effector-to-Target ratio; NK = natural killer; PBMC = peripheral blood mononuclear cell; MS = multiple sclerosis.

[0021] Figures 6A-6C provide flow cytometry -based cytotoxicity assay with PBMCs and isolated T and B cells from healthy donors. Cytotoxic activity of 3 lots of P-CD19CD20-ALLO1 CAR-T cells against PBMC samples (Figure 6A) as well as isolated T cells (Figure 6B) and B cells (Figure 6C) from three healthy donors at E:T ratios of 1:1 and 5:1 was evaluated by flow cytometry after 48 hours co-culture. Each cell population in the PBMC sample was quantified using Countbright™ beads and numbers were normalized to the PBMC samples alone without CAR-T cells. Abbreviations: CAR-T = chimeric antigen receptor T cell; E:T = Effector-to-Target ratio; HD = Healthy donor; NK = natural killer; PBMC = peripheral blood mononuclear cell.

[0022] Figure 7 provides flow cytometry-based cytotoxicity assays of CD20-positive T cells present in PBMC samples from MS patients. Cytotoxic activity of 3 lots of P-CD19CD20-ALLO1 CAR-T cells against CD20-positive T cells in PBMC samples from MS patients at E:T ratios of 1 : 1 and 5:1 was evaluated by flow cytometry after 48 hours co-culture. CD20-positive T cells within PBMC samples after treatment with the indicated CAR-T cells were quantified using Countbright™ beads and numbers were normalized to PBMC samples alone (no CAR-T) to express data as % of surviving cells after treatment with CAR-T. Only patient PBMC samples containing at least 1000 CD20-positive T cells per well were included in this assessment. Abbreviations: CAR = chimeric antigen receptor; E:T = Effector to target; MS = Multiple Sclerosis.Attorney Docket No.: 000218-0094-W01

[0023] Figures 8A and 8B provide graphs showing the relative fold change of viable P-CD19CD20-ALLO1 cells after 24 and 48 hours of treatment with rimiducid (API 903) relative to the untreated control (0 nM). Data are the average of triplicate wells with error bars presented by mean ± standard deviation.

[0024] Figures 9A and 9B provide graphs showing CD 19 and CD20 expression on target cells assessed by flow cytometry (Figure 9A) and specific cytotoxicity of P-CD19CD20-ALLO1 CAR-T cells against CD19-expressing cells and CD20-expressing cells after 72-hour coculture at 1:1 Effector to Target (E:T) ratio (Figure 9B).

[0025] Figures 10A and 10B provide graphs showing CAR expression (Figure 10A) and CAR density (Figure 10B) in dual CAR constructs compared with single CAR constructs in rested and phorbol ester + ionomycin (PMA / I)-stimulated cells evaluated by flow cytometry. Abbreviations: CAR = chimeric antigen receptor; gMFI = geometric mean fluorescence intensity.

[0026] Figures 11A-11J provide graphs showing the in vitro potency of P-CD19CD20-ALLO1 compared to its CD 19- and CD20-single targeting counterparts using serial restimulation against the CD 19- and CD20-positive (WT) RAJI cell line, as well as RAJI cells engineered to express only CD19 or CD20. Figures 11A-11C show RAJI cell killing in WT, CD19KO or CD20KO RAJI cell lines, respectively. Figures 1 ID - 11 J show the induction of secretion of the cytokines IL-2 (Figure 1 ID), TNFa (Figure HE), IFNy (Figure 1 IF), Granzyme A (Figure 11G), Granzyme B (Figure 11H), perforin (Figure 111) and Granulysin (Figure 11 J). Abbreviations: E:T = Effector to target.

[0027] Figure 12 provides a schema for a Phase I clinical trial to evaluate the safety, tolerability, pharmacokinetics (PK), cellular kinetics (CK), pharmacodynamics (PD), and efficacy of CD19CD20-ALLO1 CAR- T cells in patients with multiple sclerosis, showing patients enrolled in a dose-escalation stage and details on this stage. Each dose-escalation cohort will include a total of 6 participants with up to 6 additional participants in Cohorts 1 and 2 and up to an additional 12 participants in optional Cohorts 3 and 4. Optional Cohorts 3 and 4 may be opened based on emerging data. The initial participant receiving P-CD19CD20-ALLO1 in each cohort will be observed for a minimum of 28 days before additional participants are treated in the same cohort.Attorney Docket No.: 000218-0094-W01Subsequent participants will be staggered such that their P-CD19CD20-ALLO1 is administered at least 14 days apart. There is a dose-limiting toxicity (DLT) window of 28 days.aLymphodepletion dose of fludarabine / cyclophosphamide at 30 / 500 mg / m2x 3 days.bAlternative P-CD19CD20-ALLO1 dose / LDC regimen may be explored in optional cohorts.

[0028] Figure 13 provides the dosing and visit schedule for the CD19CD20-ALLO1 CAR- T clinical trial.aAfter discharge, participants must remain within a 1-hour travel radius from the site through Day 29.bAfter 5 years, participants will be transitioned to a separate protocol for continued monitoring for up to 15 years after P-CD19CD20-ALLO1 infusion. [DLT = dose-limiting toxicity; E = enrollment],DETAILED DESCRIPTIONGeneral

[0029] Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature.

[0030] The term “herein” means the entire application.

[0031] It should be understood that any of the embodiments described herein, including those described under different aspects of the disclosure and different parts of the specification (including embodiments described only in the Examples) can be combined with one or more other embodiments disclosed herein, unless explicitly disclaimed or improper. Combination of embodiments are not limited to those specific combinations claimed via the multiple dependent claims.

[0032] Any publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0033] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.Attorney Docket No.: 000218-0094-W01

[0034] Throughout the specification, where compositions are described as having, including, or comprising (or variations thereof), specific components, it is contemplated that compositions also may consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also may consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0035] The term “consisting of’ excludes any element, step, or ingredient not specifically recited.

[0036] The term “consisting essentially of’ limits the scope of a disclosure to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the disclosure.

[0037] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0038] The articles “a,” “an” and “the” are used herein to refer to one or to more than one ( / .< ., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0039] As used herein, the term “about” modifying the quantity of an ingredient, parameter, calculation, or measurement in the compositions employed in the methods of the disclosure refers to the variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making isolated polypeptides or pharmaceutical compositions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like without having a substantial effect on the chemical or physical attributes of the compositions or methods of the disclosure. Such variation can be within an order of magnitude, typically within 10%, more typically still within 5%, of a given value or range. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about,” the paragraphs include equivalents to the quantities. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. ForAttorney Docket No.: 000218-0094-W01example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.

[0040] The term “or” as used herein should be understood to mean “and / or,” unless the context clearly indicates otherwise.

[0041] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. The disclosure of a range should also be considered as disclosure of the endpoints of that range.

[0042] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application. The materials, methods, and examples are illustrative only and not intended to be limiting.Definitions

[0043] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0044] As used herein, the term “ablated” refers to a genetic engineering technique that involves the alteration or deletion of genetic sequence information to silence a gene or make it “null” ( / .< ., a DNA sequence variation that results in no gene product). The RNA or protein encoded by a null gene is either absent or not detectable at the molecular level or does not function at the phenotypic level.

[0045] “Administering” or “administration of’ a substance, a compound or an agent to a subject refers to the contact of that substance, compound or agent to the subject or a cell, tissue, organ or bodily fluid of the subject. For example, a compound or an agent can be administered intravenously. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. In some embodiments, the administration includes both direct administration, includingAttorney Docket No.: 000218-0094-W01self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a subject to self-administer a drug, or to have the drug administered by another and / or who provides a subject with a prescription for a drug is administering the drug to the subject.

[0046] The term “amino acid,” as used herein, refers to one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.

[0047] As used herein, the term “antibody” or “Ab” refers to an immunoglobulin molecule capable of recognizing and binding to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” can encompass any type of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, human antibodies, engineered antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single-chain antibodies, artificially selected antibodies, CDR-granted antibodies, etc.) that specifically bind to a given antigen.

[0048] As used herein, the term “antigen-binding fragment” refers to a portion (or fragment) of an antibody that retains the antibodies binding specificity. Accordingly, as used herein, an antigen-binding fragment retains the six CDRs of the reference antibody. In some embodiments, the antigen-binding fragment includes a VHH antibody. In such cases, the antigen-binding fragment only includes the three heavy chain CDRs of the reference antibody.

[0049] The terms “chimeric antigen receptor” and “CAR” are used interchangeably herein and refer to a receptor protein that has been engineered to target a specific antigen and, upon binding the antigen, activate an immune cell, such as a T cell. The chimeric receptors combine both antigen-binding and T cell activating functions into a single receptor. A CAR typically comprises (a) an ectodomain comprising an antigen recognition region; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.

[0050] As used herein the term “multiple sclerosis” or “MS” refers to an autoimmune disease of the CNS characterized by chronic inflammation, demyelination, and progressive neurologic disability.

[0051] The terms “nucleic acid,” “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For theAttorney Docket No.: 000218-0094-W01purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer.

[0052] As used herein, the term “P-CD19CD20-ALLO1” CAR-T cells refers to specific healthy donor-derived allogeneic CAR T cells that targets both CD 19 and CD20 antigens. More details regarding specific genetic modification introduced into P-CD19CD20-ALLO1 CAR-T cells are provided infra.

[0053] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. Expression of a protein in a cell can result from delivery of the protein to the cell or by delivery of a polynucleotide encoding the protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are known in the art.

[0054] As used herein, the term “relapse” in the context of MS refers to the occurrence of new or worsening neurologic symptoms attributable to MS and immediately preceded by a relatively stable or improving neurologic state of at least 30 days. Symptoms must persist for >24 hours and should not be attributable to confounding clinical factors (e.g., fever, infection, injury, adverse reactions to concomitant medications). The new or worsening neurologic symptoms must be accompanied by objective neurologic worsening consistent with an increase of at least one of the following: (i) Half a step (0.5 point) on the Expanded Disability Status Scale (EDSS); (ii) 2 points on one of the selected Functional Systems Score (FSS); or 1 point on 2 or more of the selected FSS. The change must affect the following selected FSS: pyramidal, ambulation, cerebellar, brainstem, sensory, or visual. Episodic spasms, sexual dysfunction, fatigue, mood change, or bladder or bowel urgency or incontinence will not suffice to establish a relapse.

[0055] The terms “therapeutically effective amount” and “effective amount” are used interchangeably herein an refer to that amount of the therapeutic agent being administered, as a single agent or in combination with one or more additional agents, which will relieve to some extent one or more of the symptoms of the condition being treated. In some embodiments, the therapeutically effective amount is an amount sufficient to effect beneficial or desired clinical results. With respect to the treatment of MS, a therapeutically effective amount refers to that amount which has at least one of the following effects: reduction a symptom of MS, elimination of a symptom of MS,Attorney Docket No.: 000218-0094-W01remission, reduction of a biomarker of MS, elimination of a biomarker of MS, or improvement in a clinical disease activity assessment. Non-limiting examples of common MS symptoms include fatigue; motor and coordination impairments (e.g., walking ability, gait abnormalities, and limb incoordination); muscle stiffness; spasticity; paralysis; sensory disturbances; visual problems; trouble swallowing; cognitive changes; difficulty with speech; mood changes; sexual dysfunction; and incontinence. Non-limiting examples of biomarkers include cytokines associated with B cell function and activation (e.g., B cell maturation antigen (BCMA), B-Cell activating factor (BAFF) and oligoclonal bands), autoantibodies associated with MS, T-cell subsets (e.g., activation and proliferation), and T cell engraftment (IL-7 and IL-15), biomarkers associated with neuroinflammation or neurodegeneration (e.g., Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). Non-limiting examples of clinical disease activity assessments include the Expanded Disability Status Scale (EDSS), the Nine-Hole Peg Test (9-HPT), the Timed 25-Foot Walk Test (T25FWT), the Symbol Digit Modalities Test (SDMT), the composite 12 or 24-week confirmed disability progression (12w-cCDP or 24w-cCDP), and magnetic resonance imaging (MRI). Each of these assessments is described in more detail in Example 7, infra. Additionally, the revised 2017 McDonald Criteria is used in the diagnosis of MS. See, e.g., Thompson AJ et al. Lancet Neurol. 2018;17:162-73, incorporated herein by reference in its entirety. Active SPMS is further classified as defined in Lublin FD. Eur Neurol 2014;72(Suppl l):l-5, incorporated herein by reference in its entirety. The effective amount that may be used in the present disclosure varies depending upon the manner of administration, the age, body weight, and general health of the subject. The appropriate amount and dosage regimen can be determined using routine skill in the art.

[0056] As used herein, the terms “treatment” (and grammatical variations thereof such as “treat” or “treating”) and “therapeutic treatment” refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects,Attorney Docket No.: 000218-0094-W01the CAR-T cells of the disclosure are used to delay development of a disease or to slow the progression of a disease.

[0057] As used herein, the term “refractory” to treatment in the context of multiple sclerosis (MS) refers to a condition in which the disease does not respond to one or more standard therapies. Non-limiting examples of standard therapies for MS include immunomodulators (e.g., interferons, glatiramer acetate, dimethyl fumarate, teriflunomide), cell migration modulators (e.g., fmgolimod, siponimod, natalizumab), kinase inhibitors (e.g., baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, and fenebrutinib) and cell-depleting therapies e.g., alemtuzumab, cladribine, ocrelizumab, ofatumumab). Additionally, corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone) are often administered during relapses to reduce inflammation.

[0058] The terms “variable human heavy-chain domain,” “VHH” and “nanobody” are used interchangeably herein and refer to a single antigen-binding domain derived from heavy-chain antibodies. A heavy-chain antibody is an antibody which consists only of two heavy chains and lacks the two light chains usually found in antibodies. Naturally-occurring heavy-chain antibodies can be produced by camelids, such as alpacas, llamas, camels, and vicunas. Fully human and fully humanized VHHs are known in the art. See, e.g., WO2019126574A1; and Rouet, R. et al., J. Biol. Chem, 2015, vol. 290(19): 11905-17, each of which is incorporated herein by reference in its entirety.

[0059] The terms “VCAR” and “VHH CAR” are used interchangeably herein and refer to a CAR in which the antigen recognition region comprises at least one VHH.

[0060] The sequences referenced herein are provided in Table 1, infra.Table 1: Amino acid sequences used in the methods of the present disclosure.Attorney Docket No.: 000218-0094-W01P-CD19CD20-ALLO1 CAR-T Cells

[0061] P-CD19CD20-ALLO1 CAR-T cells target both CD 19 and CD20 and have been genetically modified using an electroporation-based, non-viral (DNA transposon) gene delivery system called the piggyBac® DNA modification system.Attorney Docket No.: 000218-0094-W01

[0062] P-CD19CD20-ALLO1 CAR-T cells were manufactured from cryopreserved peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis of healthy donors. The primary attributes of P-CD19CD20-ALLO1 are conferred by the genes introduced in the PB transposon, including an anti-CD19 targeting variable human heavy-chain domain (VHH)-based CAR (VCAR) gene, an anti-CD20 targeting VCAR gene, a dihydrofolate reductase (DHFR) selection mutant protein (mutein) gene, and an inducible caspase 9 (iCasp9)-based safety switch gene. See, Figure 1.Additionally, P-CD19CD20-ALLO1 CAR-T cells are deficient for the native T-cell receptor (TCR) and human leukocyte antigen (HLA)-A, B and C major histocompatibility complex I (MHC-I) proteins, as a result of the ablation of the TCRP chain (TCRB) and p2-microglobin (P2M) genes using the Cas-CLOVER™ Site-Specific Gene Editing System. The TCRB and B2M genes are genes known to mediate rejection, e.g., graft-vs-host (GVH) and host-vs-graft (HVG) responses. Accordingly, their removal decreases the likelihood that the subject being treated will reject the P-CD19CD20-ALLO1 allogeneic CAR-T cells being administered.

[0063] P-CD19CD20-ALLO1 CAR-T cells comprise 2 CAR separate expression cassettes. The first CAR expression cassette encodes an extracellular CD 19 binding VHH protein (SEQ ID NO: 1) fused to a CD8a spacer (SEQ ID NO: 4), a CD8a transmembrane domain (SEQ ID NO: 5), an intracellular 4-1BB signaling domain (SEQ ID NO: 6) and a CD3(^ signaling domain (SEQ ID NO: 7). The second CAR expression cassette encodes an extracellular CD20-binding VHH protein (SEQ ID NO: 2) fused to a CD8a spacer (SEQ ID NO: 4), a CD8a transmembrane domain (SEQ ID NO: 5), an intracellular 4-1BB signaling domain (SEQ ID NO: 6) and a CD3(^ signaling domain (SEQ ID NO: 7). See, Figure 2 for a schematic representation of the CD19 VCAR and the CD20 VCAR. Both expression cassettes also include nucleotide sequences encoding a CD8a signal peptide (SEQ ID NO: 3), which gets cleaved from the mature VCAR in the endoplasmic reticulum.

[0064] CD 19 and CD20 are lineage-specific antigens expressed on the cell surface of certain B cells. CD 19 is expressed more broadly from early B-cell genesis through maturation and remains present on a subset of plasma cells, while CD20 is acquired during the late stage of B-cell lymphopoiesis and lost upon their differentiation into plasma cells.

[0065] Moreover, autologous CAR-T cell therapies require well-functioning T cells at apheresis from the patient. For patients who may have received previous MSAttorney Docket No.: 000218-0094-W01treatment regimens, including cell-depleting therapies, it is possible that the T-cell quality is poor at apheresis. Indeed, T cell function is adversely affected by celldepleting therapies, such as alemtuzumab and cladribine, which deplete naive or stem central memory T cells critical for CAR-T cell expansion.

[0066] P-CD19CD20-ALLO1 CAR-T cells address the issues that affect autologous CD 19 directed CAR-T therapies. Without being bound by theory, it is unlikely that treatment resistant clones will develop to a CAR-T therapy that is able to recognize two antigens simultaneously. Moreover, because P-CD19CD20-ALLO1 CAR-T cells are allogeneic, the T-cells from which they are derived are obtained from healthy donors that have never received cytotoxic chemotherapy and, therefore should result in a product with highly functional T-cells with good cell killing activity. Accordingly, P-CD19CD20-ALLO1 may achieve a greater degree of B cell depletion, including B cells in the CNS, compared with current monovalent B cell targeting antibodies preventing underlying disease progression in patients with MS.

[0067] An iCasp9 “safety switch” is an extra safety feature not found in most CAR-T cells and is designed to allow for rapid killing of P-CD19CD20-ALLO1 cells, if clinically indicated, by intravenous (IV) administration of the activation agent, rimiducid, a synthetic small molecule dimerizing drug. The iCasp9 safety switch is the first gene encoded within the quad-cistronic P-CD19CD20-ALLO1 transgene. Because it is encoded upstream of the CD 19 and CD20 VCARs, there can be no expression of the VCARs or DHFR genes without expression of the iCasp9 switch. The iCasp9 sequence consists of the chemically induced dimerization (CID)-binding domain (i.e. a mutant human FK506-binding protein 12 (FKBP12) domain) coupled to the signaling domain of caspase-9, an enzyme that is part of the apoptotic pathway. The amino acid sequence of the iCasp9 safety switch is provided in SEQ ID NO: 8 or 9. In some embodiments, the iCasp9 safety switch is encoded by the amino acid sequence of SEQ ID NO: 8. In some embodiments, the iCasp9 safety switch is encoded by the amino acid sequence of SEQ ID NO: 9. Infusion of rimiducid (AP1903), an investigational product and otherwise bio-inert small-molecule dimerizing drug, triggers dimerization of iCasp9 and subsequent activation of caspase-9, which leads to rapid induction of apoptosis of the iCasp9-containing CAR-T cells, preferentially killing activated cells expressing the transgene. Rimiducid eliminates P-CD19CD20-ALLO1 cells in a dosedependent and time-dependent manner in vitro (0.1 nM [40%], 1 nM [60%], and 10 nM [100%]).Attorney Docket No.: 000218-0094-W01

[0068] The selection gene encoding a DHFR mutein enzyme (SEQ ID NO: 10) was used during manufacturing for ex vivo selection of P-CD19CD20-ALLO1 CAR-T cells to produce a more homogeneous product. The wild-type DHFR protein plays a role in DNA synthesis is rapidly dividing cells but is inhibited by methotrexate. Accordingly, when cells are exposed to methotrexate, they eventually die. The modified DHFR selection gene used in P-CD19CD20-ALLO1 CAR-T cells, however, has a lower sensitivity to methotrexate. The modified DHFR gene is the fourth ( / .< ., last) in the quad-ci stronic P-CD19CD20-ALLO1 transgene. Accordingly, it is only expressed in cells that have expressed all of the iCasp9 safety switch, the CD 19 VCAR, and the CD20 VCAR and, therefore, can be used to select for such cells.

[0069] TCR knockout cells do not express surface TCR / CD3 complex and thus expand poorly in response to anti-CD3 agonist antibodies. To overcome this deficiency, the P-CD19CD20-ALLO1 manufacturing process uses an mRNA construct that encodes a booster molecule (CD2z booster) that is co-delivered via electroporation. CD2z mRNA encodes a chimeric fusion protein composed of human full-length CD2 and human CD3(^ intracellular signaling domain. When cells expressing the CD2z booster mRNA are treated with expansion reagents containing anti-CD2 agonist antibodies, binding of the CD2z booster rescues intracellular delivery of CD3(^ signals to the TCR knockout cells and results in optimal levels of expansion.Method of treatment of Multiple Sclerosis using P-CD19CD20-ALLO1 CAR-T Cells

[0070] The present disclosure relates to methods of treating multiple sclerosis (MS), such as progressive MS (PMS) or relapsing MS (RMS), using P-CD19CD20-ALLO1 CAR-T cells, which are allogeneic dual chimeric antigen receptor T (CAR-T) cells comprising a chimeric antigen receptor (CAR) encoding an anti-CD20 antibody and a CAR encoding an anti-CD19 antibody. Without being bound by theory, P-CD19CD20-ALLO1 CAR-T cells may achieve a greater degree of B cell depletion compared with current monovalent B cell-targeting antibodies, including more extensive loss of tissue-infiltrating B cells and more efficient removal of cells with lower levels of CD 19 or CD20 expression, which may yield improved efficacy. The hypothesized ability of CAR-T cells to provide deep depletion of tissue resident B cells that are refractory to other B cell-depleting modalities may facilitate an “immune reset” where the pathogenic B cell repertoire is eliminated, and a predominantly naive immuneAttorney Docket No.: 000218-0094-W01system is reconstituted. Therefore, P-CD19CD20-ALLO1 CAR-T cells are hypothesized to produce broad and deep depletion of autoreactive B cells, including B cells in the CNS, thus preventing underlying disease progression in patients with MS. P-CD19CD20-ALLO1 CAR-T cells are also hypothesized to deplete a subpopulation of CD20-positive T cells that have been described in some autoimmune diseases and that have a high pathogenic potential.

[0071] To confirm antigen-specific cytotoxicity against CD19-positive cells and CD20-positive cells in autoimmune samples, a series of nonclinical in vitro assays were performed to assess P-CD19CD20-ALLO1 CAR-T cell activity against primary peripheral blood mononuclear cells (PBMCs). See, Examples 2 and 3, infra. PBMCs were isolated from either healthy human donor controls or patients with autoimmune disease, including MS. Three lots of P-CD19CD20-ALLO1 CAR-T cells were manufactured from 3 individual healthy donors and characterized for in vitro cytotoxicity and cytokine release against these PBMCs. All three lots of P-CD19CD20-ALLO1 CAR-T cells demonstrated antigen-specific cytotoxicity and cytokine release against B cells and CD20-expressing T cells. Neither off-target killing nor cytokine release was observed with antigen-negative cells, with the exception of monocytes in some donors, consistent with the well-known nonspecific activation of T cells by monocytes. The data disclosed herein, therefore, demonstrate the antigenspecific potency of P-CD19CD20-ALLO1 CAR-T cells and the potential for their deployment for the treatment of MS, mediated by CD19-positive and / or CD20-positive B and T cells.

[0072] Additionally, results from a comprehensive battery of in vitro and in vivo nonclinical pharmacology and toxicology studies of P-CD19CD20-ALLO1 CAR-T cells provide evidence of target pharmacology, demonstrate an acceptable safety profile, and support administration in humans. See, Examples 4 and 5, infra. The demonstrated effects reflect the specificity of P-CD19CD20-ALLO1 CAR-T cells for both intended targets present on cell lines expressing CD 19 and / or CD20, but not for cells without these surface markers. Genotoxicity and other toxicity studies demonstrated an acceptable safety profile with no evidence of cellular transformation and with very low frequencies of off-target gene editing and translocation events. The functionality of the iCasp9 safety switch was demonstrated to be intact.Attorney Docket No.: 000218-0094-W01

[0073] In a first aspect, the present disclosure provides methods of treating multiple sclerosis (MS) in a human subject in need thereof comprising administering a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) to the subject.

[0074] In a second aspect, the present disclosure provides the use of a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) in the manufacture of a medicament for treating multiple sclerosis (MS) in a human subject in need thereof.

[0075] In a third aspect, the present disclosure provides a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) for use in treating multiple sclerosis (MS) in a human subject in need thereof.

[0076] In some embodiments of any of the above aspects, the MS is progressive MS (PMS). In some embodiments, the PMS is primary PMS (PPMS) or non-active secondary PMS (naSPMS). In some embodiments, the PMS is primary PMS (PPMS). In some embodiments, the PMS is non-active secondary PMS (naSPMS). In some embodiments, the MS is relapsing MS (RMS). In some embodiments, the RMS is relapsing remitting MS (RRMS) or active secondary PMS (aSPMS). In some embodiments, the RMS is relapsing remitting MS (RRMS). In some embodiments, the RMS is active secondary PMS (aSPMS). In some embodiments, the MS is diagnosed according to the revised 2017 McDonald Criteria. See, e.g., Thompson AJ et al. Lancet Neurol. 2018;17: 162-73, incorporated herein by reference in its entirety. Active SPMS is further classified as defined in Lublin FD. Eur Neurol 2014;72(Suppl 1): 1-5, incorporated herein by reference in its entirety. The Expanded Disability Status Scale (EDSS) is recognized in the art as an efficient assessment tool for quantifying changes in the disability level of patients with MS over time. See, e.g., Kurtzke JF. Neurology.1983; 33:1444-52 and Kappos L. Neurostatus Scoring Definitions. 2011, incorporated herein by reference in their entireties; see also, Example 7, infra. The Nine-Hole Peg Test (9-HPT) is recognized in the art as a performance measure used to assess upper extremity (arm and hand) function in patients with MS. See, e.g., Goodkin DE, et al. Arch Phys Med Rehabil. 1988;69:850-54 and Fischer, et al. Mult Scler. 1999;5:244-50, incorporated herein by reference in their entireties; see also, Example 7, infra. The Timed 25-Foot Walk Test (T25FWT) test, as described in the Multiple Sclerosis Functional Composite (MSFC) Administration and Scoring Manual, is recognized in the art as a performance measure used to assess walking speed based on a timed 25-foot walk in patients with MS. See, e.g., Fischer JS, et al. Multiple Sclerosis FunctionalAttorney Docket No.: 000218-0094-W01Composite (MSFC), Administration and Scoring Manual [resource on the Internet], October 2001, incorporated herein by reference in its entirety; see also, Example 7, infra. The Symbol Digit Modalities Test (SDMT) is recognized in the art as a performance measure that has demonstrated sensitivity in detecting the presence of cognitive impairment and changes in cognitive functioning over time and in response to treatment in patients with MS. See, e.g., Smith A. Los Angeles: Western Psychological Services, 1982 and Benedict RHB, et al. Mult Scler 2017;23:721-33, incorporated herein by reference in its entirety; see also, Example 7, infra.

[0077] In some embodiments of any of the above aspects, the CAR-T cell is administered to the subject intravenously or is formulated to be administered to the subject intravenously. In some embodiments, the CAR-T cell is administered to the subject intravenously. In some embodiments, the CAR-T cell is formulated to be administered to the subject intravenously. In some embodiments of any of the above aspects, the CAR-T cell is administered as a single dose or is formulated to be administered as a single dose. In some embodiments, the CAR-T cell is administered as a single dose. In some embodiments, the CAR-T cell is formulated to be administered as a single dose. In some embodiments, one or more subsequent doses of the CAR-T cell is administered to the subject. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 3 to 5 years after the first dose. For example, the second dose is administered 3 to 5 years after the first dose, the third dose is administered 3 to 5 years after the second dose, and so on. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 3 years after the first dose. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 3.5 years after the first dose. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 4 years after the first dose. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 4.5 years after the first dose. In some embodiments, the one or more subsequent doses of the CAR-T cell are administered to the subject every 5 years after the first dose.

[0078] In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 5 x 106and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106Attorney Docket No.: 000218-0094-W01and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106and about 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106and about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106and about 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106and about 160 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 20 x 106and about 60 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 60 x 106and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 60 x 106and about 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 60 x 106and about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 160 x 106and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 160 x 106and about 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 160 x 106and about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 320 x 106and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 320 x 106and about 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 320 x 106and about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 60 x 106and about 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 60 x 106and about 160 x 106Attorney Docket No.: 000218-0094-W01cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 160 x 106and about 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 480 x 106and about 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 480 x 106and about 800 x 106cells.

[0079] In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 5 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 20 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 60 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 160 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 1.2 x 109cells.

[0080] In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 5 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 160 x 106cells. In someAttorney Docket No.: 000218-0094-W01embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 20 x 106and 60 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 60 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 60 x 106and 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 60 x 106and 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 160 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 160 x 106and 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 160 x 106and 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 320 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 320 x 106and 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 320 x 106and 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 60 x 106and 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 60 x 106and 160 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 160 x 106and 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 480 x 106and 1.2 x 109cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 480 x 106and 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 5 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 20 x 106cells. In some embodiments of any of the above aspects, theAttorney Docket No.: 000218-0094-W01CAR-T cell is administered or is formulated to be administered at a dose of 60 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 160 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 320 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 480 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 800 x 106cells. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 1.2 x 109cells.

[0081] In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.0625 x 106and about 0.25 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.25 x 106and about 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.75 x 106and about 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 2 x 106and about 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 6 x 106and about 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 10 x 106and about 15 x 106cells / kg. In some embodiments ofAttorney Docket No.: 000218-0094-W01any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.25 x 106and about 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.25 x 106and about 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.25 x 106and about 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.25 x 106and about 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.75 x 106and about 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 0.75 x 106and about 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between about 2 x 106and about 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 0.0625 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 0.25 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of about 15 x 106cells / kg.

[0082] In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.0625 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.0625 x 106and 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cellAttorney Docket No.: 000218-0094-W01is administered or is formulated to be administered at a dose between 0.0625 x 106and 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.0625 x 106and 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.0625 x 106and 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.0625 x 106and 0.25 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.25 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.75 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 2 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 6 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 10 x 106and 15 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.25 x 106and 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.25 x 106and 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.25 x 106and 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.25 x 106and 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.75 x 106and 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 0.75 x 106and 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose between 2 x 106and 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 0.0625 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or isAttorney Docket No.: 000218-0094-W01formulated to be administered at a dose of 0.25 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 0.75 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 2 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 6 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 10 x 106cells / kg. In some embodiments of any of the above aspects, the CAR-T cell is administered or is formulated to be administered at a dose of 15 x 106cells / kg.

[0083] In some embodiments of any of the above aspects, administration of the CAR-T cell results in (a) a detectable number of CAR transgene copies in the blood of the subject, (b) a detectable number CAR-T cells in the blood of the subject, (c) the depletion of B-cells in the blood or cerebrospinal fluid (CSF) of the subject by more than 90 percent as compared to prior to treatment, (d) a reduction in the number of gadolinium lesion counts and no new or enlarging T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment, (e) no disability progression as compared to prior to treatment measured by the confirmed composite disability progression (cCDP) score confirmed at 12 or 24 weeks (24w-cCDP or 12w-cCDP) after treatment, or (f) an improvement in MS-associated biomarkers (e.g., oligoclonal bands, NfL, and GFAP) as compared to prior to treatment.

[0084] In some embodiments of any of the above aspects, administration of the CAR-T cell results in a detectable number of CAR transgene copies in the blood of the subject. In some embodiments, the number of CAR transgene copies are measured by droplet digital polymerase chain reaction (ddPCR), which is known in the art to provide accurate and precise detection of vector copy number in CAR T cell products. See, e.g, Murphy LA, et al. Cytotherapy, 2023; vol. 25(l):94-102, incorporated herein by reference in its entirety.

[0085] In some embodiments of any of the above aspects, administration of the CAR-T cell results in a detectable number CAR-T cells in the blood of the subject.

[0086] In some embodiments of any of the above aspects, administration of the CAR-T cell results in the depletion of B-cells in the blood or cerebrospinal fluid (CSF) of the subject by more than 90 percent. In some embodiments, administration of the CAR-T cell results in the depletion of B-cells in the blood of the subject. In someAttorney Docket No.: 000218-0094-W01embodiments, administration of the CAR-T cell results in the depletion of B-cells in the cerebrospinal fluid (CSF) of the subject. In some embodiments, the B-cell are depleted by more than 90 percent. In some embodiment, the B-cells are depleted in the blood of the subject by more than 95 percent. In some embodiment, the B-cells are depleted in the CSF of the subject by more than 95 percent. In some embodiment, the B-cells are depleted in the blood of the subject by more than 97 percent. In some embodiment, the B-cells are depleted in the CSF of the subject by more than 97 percent. In some embodiment, the B-cells are depleted in the blood of the subject by more than 99 percent. In some embodiment, the B-cells are depleted in the CSF of the subject by more than 99 percent. In some embodiments, after administration of the CAR-T cells, the number of B-cells in the blood or cerebrospinal fluid (CSF) of the subject falls below the limit of detection by flow cytometry. In some embodiments, after administration of the CAR-T cells, the number of B-cells in the blood of the subject falls below the limit of detection by flow cytometry. In some embodiments, after administration of the CAR-T cells, the number of B-cells in the cerebrospinal fluid (CSF) of the subject falls below the limit of detection by flow cytometry. In some embodiments, depletion of the number of B-cells occurs within 1 month after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs within 2 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs within 3 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs within 4 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs within 5 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs within 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 1 and 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 2 and 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 3 and 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 4 and 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 5 and 6 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 1 and 2 months after administration of the CAR-T cell. In someAttorney Docket No.: 000218-0094-W01embodiments, depletion of the number of B-cells occurs between 1 and 3 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 1 and 4 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 1 and 5 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 2 and 5 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 2 and 4 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 2 and 3 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 3 and 5 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 3 and 4 months after administration of the CAR-T cell. In some embodiments, depletion of the number of B-cells occurs between 4 and 5 months after administration of the CAR-T cell.

[0087] Without being bound by theory, the ability of P-CD19CD20-ALLO1 CAR-T cells to provide depletion of B cells that are refractory to other B cell-depleting modalities may facilitate an “immune reset” where the pathogenic B cell repertoire is eliminated, and a predominantly naive immune system is reconstituted. In some embodiments, a naive B-cell population is reconstituted in the subject after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 6 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 7 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 8 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 9 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 10 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 11 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject about 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 6 and 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 6 and 11 months after administration of the CAR-T cell. In someAttorney Docket No.: 000218-0094-W01embodiments, a naive B-cell population is reconstituted in the subject between 6 and 10 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 6 and 9 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 6 and 8 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 6 and 7 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 7 and 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 7 and 11 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 7 and 10 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 7 and 9 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 7 and 8 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 8 and 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 8 and 11 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 8 and 10 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 8 and 9 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 9 and 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 9 and 11 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 9 and 10 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 10 and 12 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 10 and 11 months after administration of the CAR-T cell. In some embodiments, a naive B-cell population is reconstituted in the subject between 11 and 12 months after administration of the CAR-T cell.Attorney Docket No.: 000218-0094-W01

[0088] In some embodiments of any of the above aspects, administration of the CAR-T cell results in a reduction in the number of gadolinium lesion counts and no new or enlarging T2 lesion counts by magnetic resonance imaging (MRI) as compared to prior to treatment. In some embodiments, administration of the CAR-T cell results in a reduction in the number of gadolinium lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment. In some embodiments, administration of the CAR-T cell results in no new T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment. In some embodiments, administration of the CAR-T cell results in no enlarging T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment.

[0089] In some embodiments, administration of the CAR-T cell results in no disability progression in the subject. In some embodiments, disability progression is measured by the composite disability progression score confirmed at 12 or 24 weeks (24w-cCDP or 12w-cCDP) after administration of the CAR-T cell.

[0090] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an improvement in MS-associated biomarkers (e.g., oligoclonal bands, Neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP), as compared to prior to treatment. In some embodiments, the MS-associated biomarkers comprise one or more of oligoclonal bands, NfL, and GFAP. In some embodiments, the MS-associated biomarkers comprise oligoclonal bands. In some embodiments, the MS-associated biomarkers comprise NfL. In some embodiments, the MS-associated biomarkers comprise GFAP.

[0091] In some embodiments of any of the above aspects, administration of the CAR-T cell results in (a) an incidence of Grade 2 cytokine release syndrome (CRS) less than 30%, (b) an incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) less than 20%, (c) an incidence of hemophagocytic lymphohistiocytosis (HLH) less than 10%, (d) a risk of serious infection no more than 10% more compared to the incidence of infection resulting from standard of care 6 months after administration of the CAR-T cell, (e) an incidence of immune rejection and graft-versus-host disease (GVHD) less than 10%, (f) an incidence of progressive multifocal leukoencephalopathy (PML) less than 10%, (g) an incidence of CAR T cell-driven worsening of MS in less than 20% of subjects administered the CAR-T cell, or (i) an incidence of new T-cell malignancies less than 0.1%.Attorney Docket No.: 000218-0094-W01

[0092] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of Grade 2 cytokine release syndrome (CRS) less than 30 percent. In some embodiments, the incidence if Grade 2 CRS is less than 25 percent. In some embodiments, the incidence if Grade 2 CRS is less than 20 percent. In some embodiments, the incidence if Grade 2 CRS is less than 15 percent. In some embodiments, the incidence if Grade 2 CRS is less than 10 percent. In some embodiments, the incidence if Grade 2 CRS is less than 5 percent. As used herein, CRS is assessed according to the American Society for Transplantation and Cellular Therapy (ASTCT) Consensus CRS Grading. See, e.g., Lee, DW et al., Biol Blood Marrow Transplant. 2019 Apr;25(4):625-638, incorporated herein by reference in its entirety.

[0093] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) less than 20 percent. In some embodiments, the incidence of ICANS is less than 15 percent. In some embodiments, the incidence of ICANS is less than 10 percent. In some embodiments, the incidence of ICANS is less than 5 percent. As used herein, ICANS is assessed according to ASTCT Consensus ICANS Grading. See, e.g., Lee, DW et al., Biol Blood Marrow Transplant. 2019 Apr;25(4):625-638, incorporated herein by reference in its entirety.

[0094] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of hemophagocytic lymphohistiocytosis (HLH) less than 10 percent. In some embodiments, the incidence of HLH is between 5 and 10 percent. In some embodiments, the incidence of HLH is less than 5 percent. As used herein, HLH is assessed according to ASTCT Consensus HLH Grading. See, e.g., Hines, MR. et al., Transplantation and Cellular Therapy, 2023, vol.29(7): 438. el -438. el 6, incorporated herein by reference in its entirety.

[0095] In some embodiments of any of the above aspects, administration of the CAR-T cell results in a risk of serious infection no more than 10 percent more compared to the incidence of serious infection in patients receiving standard of care at 6 months after treatment. In some embodiments, the risk of serious infection no more than 5 percent more compared to the incidence of serious infection in patients receiving standard of care at 6 months after treatment. In some embodiments, the risk of serious infection is similar to the incidence of serious infection in patients receiving standard of care at 6 months after treatment. The incidence of serious infection in patients receiving standard of care is known in the art. See, e.g., Rodziewicz, M et al. TheAttorney Docket No.: 000218-0094-W01Lancet Rheumatology, 2023, Volume 5, Issue 5, e284-e292, incorporated herein by reference in its entirety.

[0096] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of immune rejection and graft-versus-host disease (GVHD) less than 10 percent. As used herein, GVHD will be assessed according to the International Bone Marrow Transplant Registry (IBMTR) criteria. See, e.g., Rowlings, PA. et al. British J. of Haematology, 1997, vol. 97(4): 855-864, incorporated herein by reference in its entirety.

[0097] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of progressive multifocal leukoencephalopathy (PML) less than 10 percent. In some embodiments, the incidence of PML is between 5 and 10 percent. In some embodiments, the incidence of PML is less than 5 percent. As used herein, patients are screened for signs and symptoms of PML through evaluation of neurologic deficits localized to the cerebral cortex, such as cortical symptoms or signs, behavioral and neuropsychologic alteration, retrochiasmal visual defects, hemiparesis, and cerebellar symptoms or signs (e.g., gait abnormalities and limb incoordination. A clinical presentation suggestive of PML, necessitates further investigations including MRI, preferably with contrast (e.g., gadolinium), as soon as possible. In patients with an MRI evaluation revealing lesions suspicious for PML, a lumbar puncture will be performed to evaluate the CSF for the detection of John Cunningham virus (JCV) DNA using a validated sensitive assay to confirm the diagnosis of PML. The diagnostic criteria for PML are known in the art. See, e.g., Berger JR et al. Neurology, 2013, 80(15): 1430-1438, incorporated herein by reference in its entirety.

[0098] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of CAR T cell-driven worsening of MS in less than 20 percent of subjects administered the CAR-T cell. In some embodiments, the administration of the CAR T cell results in an incidence of CAR T cell-driven worsening of MS in less than 15 percent of subjects administered the CAR-T cell. In some embodiments, administration of the CAR-T cell results in an incidence of CAR T cell-driven worsening of MS in less than 10 percent of subjects administered the CAR-T cell. In some embodiments, administration of the CAR-T cell results in an incidence of CAR T cell-driven worsening of MS in less than 5 percent of subjects administered the CAR-T cell.Attorney Docket No.: 000218-0094-W01

[0099] In some embodiments of any of the above aspects, administration of the CAR-T cell results in an incidence of new T-cell malignancies less than 0.1%.

[0100] In some embodiments of any of the above aspects, the subject has not been previously administered a therapeutic agent to treat MS. In some embodiments of any of the above aspects, the subject has been previously administered one or more therapeutic agents to treat MS. In some embodiments, the subject is refractory to the one or more previously administered therapeutic agents. In some embodiments, the one or more previously administered therapeutic agents is selected from an immunomodulator, a cell migration modulator, a lymphocyte depleting therapy, a kinase inhibitor, or a corticosteroid. In some embodiments, one or more of the previously administered therapeutic agents is an immunomodulator. In some embodiments, the immunomodulator is selected from the group consisting of interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, teriflunomide, and Mitoxantrone. In some embodiments, one or more of the previously administered therapeutic agents is a cell migration modulator. In some embodiments, the cell migration modulator is selected from the group consisting of fmgolimod, siponimod, ozanimod, and natalizumab. In some embodiments, one or more of the previously administered therapeutic agents is a lymphocyte depleting therapy. In some embodiments, the lymphocyte depleting therapy is selected from the group consisting of alemtuzumab, cladribine, ocrelizumab, rituximab, ublituximab, and ofatumumab. In some embodiments, one or more of the previously administered therapeutic agents is a kinase inhibitor. In some embodiments, the kinase inhibitor is selected from the group consisting of baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, and fenebrutinib. In some embodiments, one or more of the previously administered therapeutic agents is a corticosteroid. In some embodiments, the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.

[0101] In some embodiments of any of the above aspects, the CAR-T cell is administered or formulated to be administered in combination with one or more additional therapeutic agents. In some embodiments, the CAR-T cell is administered or formulated to be administered sequentially with the one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents is selected from an immunosuppressant, a corticosteroid, a kinase inhibitor, a cytokine blocker, or a combination thereof. In some embodiments, the one or more additionalAttorney Docket No.: 000218-0094-W01therapeutic agents is an immunosuppressant. In some embodiments, the immunosuppressant is selected from the group consisting of cyclophosphamide, tocilizumab, siltuximab and anakinra. In some embodiments, the one or more additional therapeutic agents is a corticosteroid. In some embodiments, the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone. In some embodiments, the one or more additional therapeutic agents is a kinase inhibitor. In some embodiments, the kinase inhibitor is ruxolitinib. In some embodiments, the one or more additional therapeutic agents is a cytokine blocker. In some embodiments, the cytokine blocker is selected from the group consisting of ruxolitinib, etoposide, or emapalumab.

[0102] In some embodiments of any of the above aspects, the subject has been treated with a lymphodepleting chemotherapy (LDC) regimen prior to the administration of CAR-T cell. In some embodiments, the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) and fludarabine (0-30 mg / m2IV) for 3 consecutive days. In some embodiments, the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) and fludarabine (30 mg / m2IV) for 3 consecutive days. In some embodiments, the LDC regimen comprises cyclophosphamide (500 mg / m2IV) and fludarabine (30 mg / m2IV) for 3 consecutive days. In some embodiments, the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) for 3 consecutive days. In some embodiments, the LDC regimen is administered on Days -5, -4, and -3, and the CAR-T cells is administered or to be administered on Day 1.Articles of Manufacture

[0103] A fourth aspect of the present disclosure provides a dose infusion bag comprising a composition comprising P-CD19CD20-ALLO1 CAR-T cells. In some embodiments, the dose infusion bag is configured from intravenous administration.

[0104] In some embodiments, the composition comprises from about 5 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 20 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 20 x 106and about 800 x 106cells. In some embodiments, the composition comprises from about 20 x 106cells to about 480 x 106cells. In some embodiments, the composition comprises from about 20 x 106cells to about 320 x 106cells. In some embodiments, the composition comprises from about 20 x 106cells to about 160 x 106Attorney Docket No.: 000218-0094-W01cells. In some embodiments, the composition comprises from about 20 x 106cells to about 60 x 106cells. In some embodiments, the composition comprises from about 60 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 60 x 106and about 800 x 106cells. In some embodiments, the composition comprises from about 60 x 106cells to about 480 x 106cells. In some embodiments, the composition comprises from about 160 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 160 x 106and about 800 x 106cells. In some embodiments, the composition comprises from about 160 x 106cells to about 480 x 106cells. In some embodiments, the composition comprises from about 320 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 320 x 106and about 800 x 106cells. In some embodiments, the composition comprises from about 320 x 106cells to about 480 x 106cells. In some embodiments, the composition comprises from about 60 x 106cells to about 320 x 106cells. In some embodiments, the composition comprises from about 60 x 106cells to about 160 x 106cells. In some embodiments, the composition comprises from about 160 x 106cells to about 320 x 106cells. In some embodiments, the composition comprises from about 480 x 106and about 1.2 x 109cells. In some embodiments, the composition comprises from about 480 x 106and about 800 x 106cells. In some embodiments, the composition comprises about 5 x 106cells. In some embodiments, the composition comprises about 20 x 106cells. In some embodiments, the composition comprises about 60 x 106cells. In some embodiments, the composition comprises about 160 x 106cells. In some embodiments, the composition comprises about 320 x 106cells. In some embodiments, the composition comprises about 480 x lO6cells. In some embodiments, the composition comprises about 800 x 106cells. In some embodiments, the composition comprises about 1.2 x 109cells.

[0105] In some embodiments, the composition comprises from 5 x 106and 1.2 x 109cells. In some embodiments, the composition comprises from 20 x 106and 1.2 x 109cells. In some embodiments, the composition comprises from 20 x 106and 800 x 106cells. In some embodiments, the composition comprises from 20 x 106cells to 480 x 106cells. In some embodiments, the composition comprises from 20 x 106cells to 320 x lO6cells. In some embodiments, the composition comprises from 20 x 106cells to 160 x 106cells. In some embodiments, the composition comprises from 20 x 106cells to 60 x 106cells. In some embodiments, the composition comprises from 60 x 106and 1.2 x 109cells. In some embodiments, the composition comprisesAttorney Docket No.: 000218-0094-W01from 60 x 106and 800 x 106cells. In some embodiments, the composition comprises from 60 x l06cells to 480 x lO6cells. In some embodiments, the composition comprises from 160 x 106and 1.2 x 109cells. In some embodiments, the composition comprises from 160 x 106and 800 x 106cells. In some embodiments, the composition comprises from 160 x lO6cells to 480 x lO6cells. In some embodiments, the composition comprises from 320 x 106and 1.2 x 109cells. In some embodiments, the composition comprises from 320 x 106and 800 x 106cells. In some embodiments, the composition comprises from 320 x 106cells to 480 x 106cells. In some embodiments, the composition comprises from 60 x l06cells to 320 x lO6cells. In some embodiments, the composition comprises from 60 x 106cells to 160 x 106cells. In some embodiments, the composition comprises from 160 x 106cells to 320 x 106cells. In some embodiments, the composition comprises from 480 x 106and 1.2 x 109cells. In some embodiments, the composition comprises from 480 x 106and 800 x 106cells. In some embodiments, the composition comprises 5 x 106cells. In some embodiments, the composition comprises 20 x 106cells. In some embodiments, the composition comprises 60 x 106cells. In some embodiments, the composition comprises 160 x 106cells. In some embodiments, the composition comprises 320 x 106cells. In some embodiments, the composition comprises 480 x 106cells. In some embodiments, the composition comprises 800 x lO6cells. In some embodiments, the composition comprises 1.2 x 109cells.EXEMPLARY EMBODIMENTS

[0106] Particular embodiments of the disclosure are set forth in the following numbered paragraphs:1. A method of treating multiple sclerosis (MS) in a subject in need thereof comprising administering a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (C AR-T cell) to the subj ect.2. Use of a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) in the manufacture of a medicament for treating multiple sclerosis (MS) in a subject in need thereof.3. A P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) for use in treating multiple sclerosis (MS) in a subject in need thereof.Attorney Docket No.: 000218-0094-W014. The method according to paragraph 1, the use according to paragraph 2, or the CAR-T cell for use according to paragraph 3, wherein the MS is progressive MS (PMS).5. The method, use or CAR-T for use according to paragraph 4, wherein the PMS is primary PMS (PPMS) or non-active secondary PMS (naSPMS).6. The method according to paragraph 1, the use according to paragraph 2, or the CAR-T cell for use according to paragraph 3, wherein the MS is relapsing MS (RMS).7. The method, use or CAR-T for use according to paragraph 6, wherein the RMS is relapsing remitting MS (RRMS) or active secondary progressive MS (aSPMS). 8. The method, use or CAR-T cell for use according to any one of paragraphs 4-7, wherein the MS is diagnosed according to the revised 2017 McDonald Criteria.9. The method according to any one of paragraphs 1 and 4-8, the use according to any one of paragraphs 2 and 4-8, or the CAR-T cell for use according to any one of paragraphs 3-8, wherein the CAR-T cell is administered to the subject intravenously or is formulated to be administered to the subject intravenously.10. The method according to any one of paragraphs 1 and 4-9, the use according to any one of paragraphs 2 and 4-9, or the CAR-T cell for use according to any one of paragraphs 3-9, wherein the CAR-T cell is administered as a single dose or is formulated to be administered as a single dose.11. The method according to any one of paragraphs 1 and 4-9, the use according to any one of paragraphs 2 and 4-9, or the CAR-T cell for use according to any one of paragraphs 3-9, wherein the CAR-T cell is administered in multiple doses or is formulated to be administered in multiple doses, optionally wherein each of the multiple doses is administered between 3 and 5 years after the previous dose.12. The method according to any one of paragraphs 1 and 4-11, the use according to any one of paragraphs 2 and 4-11, or the CAR-T cell for use according to any one of paragraphs 3-11, wherein the CAR-T cell is administered at a dose of about 5 x 106cells to about 1.2 x 109cells or is formulated to be administered at a dose of about 5 x 106cells to about 1.2 x 109cells.13. The method according to any one of paragraphs 1 and 4-12, the use according to any one of paragraphs 2 and 4-12, or the CAR-T cell for use according to any one of paragraphs 3-12, wherein administration of the CAR-T cell results in (a) a detectableAttorney Docket No.: 000218-0094-W01number of CAR transgene copies in the blood of the subject, (b) a detectable number of CAR-T cells in the blood of the subject, (c) the depletion of B-cells in the blood or cerebrospinal fluid (CSF) of the subject by more than 90 percent as compared to prior to treatment, (d) a reduction in the number of gadolinium lesion counts and no new or enlarging T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment, (e) no disability progression as compared to prior to treatment measured by the confirmed composite disability progression (cCDP) score confirmed at 12 or 24 weeks (24w-cCDP or 12w-cCDP) after treatment, or (f) an improvement in MS-associated biomarkers (e.g., oligoclonal bands, NfL, and GFAP) as compared to prior to treatment.14. The method according to any one of paragraphs 1 and 4-13, the use according to any one of paragraphs 2 and 4-13, or the CAR-T cell for use according to any one of paragraphs 3-13, wherein administration of the CAR-T cell results in (a) an incidence of Grade 2 cytokine release syndrome (CRS) less than 30%, (b) an incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) less than 20%, (c) an incidence of hemophagocytic lymphohistiocytosis (HLH) less than 10%, (d) a risk of serious infection no more than 10% more compared to the incidence of infection resulting from standard of care 6 months after administration of the CAR-T cell, (e) an incidence of immune rejection and graft-versus-host disease (GVHD) less than 10%, (f) an incidence of progressive multifocal leukoencephalopathy (PML) less than 10%, (g) an incidence of CAR T cell-driven worsening of MS in less than 20% of subjects administered the CAR-T cell, or (h) an incidence of new T-cell malignancies less than 0.1%.15. The method according to any one of paragraphs 1 and 4-14, the use according to any one of paragraphs 2 and 4-14, or the CAR-T cell for use according to any one of paragraphs 3-14, wherein the subject has not been previously administered a therapeutic agent to treat MS.16. The method according to any one of paragraphs 1 and 4-14, the use according to any one of paragraphs 2 and 4-14, or the CAR-T cell for use according to any one of paragraphs 3-14, wherein the subject has been previously administered one or more therapeutic agents to treat MS.Attorney Docket No.: 000218-0094-W0117. The method, use, or CAR-T cell for use according to paragraph 16, wherein the subject is refractory to the one or more previously administered therapeutic agents. 18. The method, use, or CAR-T cell for use according to paragraph 16 or 17, wherein the one or more previously administered therapeutic agents are selected from an immunomodulator, a cell migration modulator, a lymphocyte depleting therapy, a kinase inhibitor, or a corticosteroid.19. The method, use, or CAR-T cell for use according to paragraph 18, wherein the immunomodulator is selected from the group consisting of interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, teriflunomide, and mitoxantrone.20. The method, use, or CAR-T cell for use according to paragraph 18, wherein the cell migration modulator is selected from the group consisting of fmgolimod, siponimod, ozanimod, and natalizumab.21. The method, use, or CAR-T cell for use according to paragraph 18, wherein the lymphocyte depleting therapy is selected from the group consisting of alemtuzumab, cladribine, ocrelizumab, rituximab, ublituximab, and ofatumumab.22. The method, use, or CAR-T cell for use according to paragraph 18, wherein the kinase inhibitor is selected from the group consisting of baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, and fenebrutinib.23. The method, use, or CAR-T cell for use according to paragraph 18, wherein the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.24. The method according to any one of paragraphs 1 and 4-23, the use according to any one of paragraphs 2 and 4-23, or the CAR-T cell for use according to any one of paragraphs 3-23, wherein the CAR-T cell is administered or formulated to be administered in combination with one or more additional therapeutic agents.25. The method, use, or CAR-T cell for use according to paragraph 24, wherein the CAR-T cell is administered or formulated to be administered sequentially with the one or more additional therapeutic agents.Attorney Docket No.: 000218-0094-W0126. The method, use, or CAR-T cell for use according to paragraph 24 or 25, wherein the one or more additional therapeutic agents is selected from an immunosuppressant, a corticosteroid, a kinase inhibitor, or a cytokine blocker.27. The method, use, or CAR-T cell for use according to paragraph 26, wherein the immunosuppressant is selected from the group consisting of cyclophosphamide, tocilizumab, siltuximab and anakinra.28. The method, use, or CAR-T cell for use according to paragraph 26, wherein the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.29. The method, use, or CAR-T cell for use according to paragraph 26, wherein the kinase inhibitor is ruxolitinib.30. The method, use, or CAR-T cell for use according to paragraph 26, wherein the cytokine blocker is selected from the group consisting of ruxolitinib, etoposide, or emapalumab.31. The method according to any one of paragraphs 1 and 4-30, the use according to any one of paragraphs 2 and 4-30, or the CAR-T cell for use according to any one of paragraphs 3-30, wherein the subject receives a lymphodepleting chemotherapy (LDC) regimen prior to the administration of CAR-T cell.32. The method, use, or CAR-T cell for use according to paragraph 31, wherein the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) and fludarabine (0-30 mg / m2IV) for 3 consecutive days.33. The method, use, or CAR-T cell for use according to paragraph 31 or 32, wherein the LDC regimen is administered on Days -5, -4, and -3, and the CAR-T cells is administered or to be administered on Day 1.EXAMPLESExample 1: Production of P-CD19CD20-ALLO1 cells

[0107] Healthy donor PBMCs were harvested by a standard leukapheresis and cryostored at < 130°C until use. After thaw, CD4+ and CD8+ T cells were isolated from the leukapheresis material and electroporated with the Cas-CLOVER™ mRNA, the Super piggyBac® (SPB) transposase mRNA, CD2z Booster mRNA, guide RNAsAttorney Docket No.: 000218-0094-W01for P2M and TCR P chain, and PB transposon carrying the CD 19 and CD20 transgene. After ex vivo cell expansion during manufacture, followed by purification to remove TCR-positive T cells, modified T cells were formulated and cryopreserved to yield the P-CD19CD20-ALLO1 CAR-T cells.Example 2: In Vitro Primary pharmacology of P-CD19CD20-ALLO1

[0108] Primary pharmacology in vitro studies characterized P-CD19CD20-ALLO1 CAR-T cells for phenotype and target-specific activity. P-CD19CD20-ALLO1 CAR-T cells all had high viability > 91% and demonstrated consistently high knockout frequencies. All lots of P-CD19CD20-ALLO1 CAR-T cells were comprised of 43.5% to 63.9% stem cell-like memory T cells (TSCM) in CD8+ T cells. Table 2, infra, summarizes the cell surface marker expression profile, CD3 and B2M KO frequencies, CAR expression (CD19-CAR and CD20-CAR double-positive of CD4- or CD8-positive cells) and viability of P-CD19CD20-ALLO1 CAR-T products. P-CD19CD20-ALLO1 produced potent, antigen-specific cytotoxicity (Figures 3 A and 3B) and secretion of the proinflammatory cytokine interferon gamma (fFN-y) (Figures 4A and 4B) and granzyme B (Figures 4C and 4D).Table 2: Summary of Surface Marker Expression and Viability for RUO ProductionsAbbreviations: B2M = beta-2 -microglobulin; CAR = chimeric antigen receptor; CD = cluster of differentiation; KO = knockout; NC-200 = NucleoCounter®-200; TSCM = stem cell memory T cells.Example 3: In Vitro P -CD19CD20 -ALLO1 CAR-T Cell Anti-B Cell Activity Assessment

[0109] The in vitro potency activity of 3 lots of P-CD19CD20-ALLO1 CAR-T cells was evaluated on PBMC’s isolated from patients with MS. After 48 hours of co-culture, all three lots of P-CD19CD20-ALLO1 CAR-T cells consistently demonstrated antigen-specific and robust depletion of B cells (Figure 5). Overall, NK cells and T cells were minimally targeted, although we noted cytotoxic activity against a population of CD20-positive T cells present in some autoimmune disease PBMCs.Attorney Docket No.: 000218-0094-W01While monocyte depletion was noted with some PBMC samples, co-culture assays with isolated monocytes did not demonstrate significant cytotoxicity, suggesting the cytotoxicity observed in PBMCs was likely due to bystander killing.

[0110] Similar levels of antigen-specific cytotoxic activity of P-CD19CD20-ALLO1 CAR-T cells were observed in co-culture assays with HD PBMCs or isolated T or B cells (Figures 6A-6C). Cytotoxicity against B cells obtained from healthy donors and patients suffering from autoimmune disease was similar to that observed against the Raji tumor cell line, which expresses both CD19 and CD20 antigens at a higher level than normal B cells. See, Example 5, infra. The higher background targeting of NK cells, T cells and monocytes that was observed with Donor 3 in PBMCs was not observed against isolated T cells from the same donor indicating that this background is most likely due to bystander killing by activated P-CD19CD20-ALLO1 CAR-T cells rather than specific targeting. In addition to cytotoxic activity, P-CD19CD20-ALLO1 CAR-T cells also demonstrated significant cytokine release against antigen-positive B from both autoimmune patients and healthy donors, but not antigen-negative cells.

[0111] T cells in autoimmune diseases can express low levels of CD20 and have a high pathogenic potential. P-CD19CD20-ALLO1 T cells induced a strong and E:T-dependent reduction of the number of CD20-positive T cells in MS Donors 1, 2 and 4 (Figure 7), demonstrating a potential to target both pathogenic B cells and CD20-expressing T cells.Example 4: In Vitro Rimiducid (AP1903) Killing of P-CD19CD20-ALLO1 Cells Expressing Inducible Caspase-9 Protein

[0112] The ability of the P-CD19CD20-ALLO1 iCasp9 safety switch to induce cellular apoptosis in response to administration of rimiducid (AP1903) was evaluated. The iCasp9 gene, an inducible apoptosis system is incorporated into the P-CD19CD20-ALLO1 transgene to eliminate CAR-T cells after administration of another investigational drug, rimiducid (AP1903) in patients, if indicated. Upon binding to API 903, iCasp9 protein dimerizes and triggers the apoptotic caspase cascade, leading to cell death.

[0113] API 903 eliminated P-CD19CD20-ALLO1 cells in a dose-dependent and time-dependent manner in vitro; 0.1, 1 and 10 nM API 903 eliminated approximatelyAttorney Docket No.: 000218-0094-W0140%, 60%, and 100% of P-CD19CD20-ALLO1 cells by 24 hours, respectively (Figure 8 A), and approximately 40%, 65%, and 100% of P-CD19CD20-ALLO1 cells by 48 hours (Figure 8B). Activated P-CD19CD20-ALLO1 cells were more sensitive to AP1903.Example 5: In Vitro P-CD19CD20-ALLO1 Reactivity Assessment Against Normal Human Cells

[0114] The reactivity of P-CD19CD20-ALLO1 CAR-T cells was evaluated against a panel of normal human cells representative of a broad range of organs and tissue types including 12 normal primary human cells and 8 tumor cell lines along with concurrent assessment of the surface expression of CD 19 and CD20 on target cells by flow cytometry (Figure 9A).

[0115] P-CD19CD20-ALLO1 CAR-T cells specifically lysed the CD19 / CD20 double-positive tumor cell line Raji compared to tumor alone and had no nonspecific killing against the CD19 / CD20 negative cell line Jurkat. Similarly, P-CD19CD20-ALLO1 demonstrated antigen-specific cytotoxicity against all tumor cell lines that expressed CD19 and / or CD20 including against single-positive Raji CD20KO, Raji CD19KO, Jeko-1, Nalm-6, and Mec-1 cells, and the B Lymphoblast cell line COLO829BL. Of the normal human primary cells, specific cytotoxicity was only observed against bone marrow and PBMCs, both of which had a small fraction of cells that stained positive for CD 19 and CD20 by flow cytometry, as expected (Figure 9B).Example 6: In Vitro Potency Assessment of P-CD19CD20-ALLO1 compared to CD19- and CD20-Single Targeting Counterparts

[0116] The potency of P-CD19CD20-ALLO1 was compared to the potency of its CD 19- and CD20-single targeting counterparts, and potency advantages were observed. In vitro potency was evaluated using serial restimulation against the CD 19-and CD20-positive RAJI cell line, as well as RAJI cells engineered to express only CD 19 or only CD20. In these assays, P-CD19CD20-ALLO1 CAR-T cells showed superior potency against WT (CD19+CD20+) RAJI cells compared to the CD19- or CD20-single targeting products (Figure 11 A). Interestingly, this superiority was also observed against Raji cells expressing only one of the two target antigens, CD 19 or CD20 (Figures 1 IB and 11C), suggesting that the increased potency of P-CD19CD20-ALLO1 is not solely due to the dual binding of CAR molecules to more antigens on theAttorney Docket No.: 000218-0094-W01same target cell. Mechanistically, it was observed that over three restimulations with WT (CD19+CD20+) RAJI cells, P-CD19CD20-ALLO1 CAR-T cells expressed and / or sustained higher expression of effector cytokines such as IL-2, TNFa, IFNy, Granzyme A, Granzyme B, perforin and Granulysin than either CD 19 or CD20 single-antigen targeting CAR-T cells (Figures 11D-11J).

[0117] In addition to improved in vitro potency, the in vivo efficacy of the dualtargeting product in comparison with the single-targeting products was assessed in a stress xenograft model of WT (CD19+CD20+) RAJI. P-CD19CD20-ALLO1 CAR-T cells were superior to the CD19-targeting CAR-T product (data not shown). P-CD19CD20-ALLO1, a dual-targeting, fully allogeneic TSCM-rich CAR-T product for CD 19 and CD20-positive B-cell malignancies, demonstrates robust antigen-specific activity against diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) models and outperforms its single-targeting counterparts even in the presence of only a single antigen on target cells.Example 7: Open-label, multicenter study of P-CD19CD20-ALLO1 CAR-T Cells in subjects with Multiple Sclerosis

[0118] A Phase I, multi center, open-label study to evaluate the safety, tolerability, cellular kinetics (CK), and pharmacodynamics (PD) of P-CD19CD20-ALLO1 CAR-T cells will be conducted in subjects with multiple sclerosis (MS). The study will include a dose-escalation stage. An overview of the study design, including the dosing plan for each stage, is provided in Figures 12 and 13.

[0119] Approximately 18 participants (up to a maximum of 60 participants) with MS will be enrolled in 3-4 cohorts in this study, in accordance with the inclusion and exclusion criteria listed below.Inclusion Criteria:■ Signed Informed Consent Form.■ Age 18-60 years (inclusive) at time of signing Informed Consent Form.■ Ability to comply with the study protocol, including remaining within an approximately 1-hour travel radius of the treatment site for 28 days after P- CD19CD20-ALLO1 infusion.■ Sufficient proficiency in the language of the investigator and study staff, or ability to communicate with the study staff (e.g., using a qualified interpreter).■ Agreement to adhere to the contraception requirements.Attorney Docket No.: 000218-0094-W01■ For patients with progressive forms of MS (PMS): Cohorts 1 and 2 and Optional Cohorts (if applicable):o Diagnosis of progressive MS (non-active SPMS or PPMS) according to the revised McDonald 2017 criteria (Thompson et al.2018).o Expanded disability status scale (EDSS) score at screening, from 3 to 6 inclusive.o Documented EDSS progression of > 1 point in the 2 years prior to screening. A written summary of the clinical evidence of disability progression in the 2 years prior to screening is also acceptable. o No evidence of relapses within 24 months of screening ■ For patients with relapsing forms of MS (RMS): Optional Cohorts (if applicable)o Diagnosis of RMS (may include active SPMS as defined by Lublin

[2014] ) in accordance with the revised 2017 McDonald Criteria (Thompson et al. 2018)o Both of the following (while on a DMT for a clinically relevant period of time, and with an appropriate dose of the DMT):■ At least two documented clinical relapses within the last 2 years prior to screening, or one clinical relapse in the year prior to screening■ Evidence of MRI activity: one or more T1 Gd-enhancing lesions and / or one or more new / enlarging T2 lesions. Evidence for Gd-enhancing lesions should be present in the screening MRI or in an MRI acquired within 12 months prior to screening. Evidence for new / enlarging T2 lesions should be based on the comparison of two MRI scans: the first MRI should be within 1 year of the second; the second MRI can either be an existing MRI performed within 1 year of screening, or an MRI performed during the screening period using the study scanning protocol.o EDSS score at screening, from 0 to 6 inclusive.Attorney Docket No.: 000218-0094-W01o No relapse for 45 days prior to screening and neurologically stable with no relapse between screening and the day before the initiation of LDC therapy (Day -6).■ Participants requiring symptomatic treatment for MS (e.g., fampridine, cannabis) and / or physiotherapy must be treated at a stable dose during the screening period prior to the initiation of LDC therapy (Day -5) and must have a plan to remain at a stable dose throughout the first year of the study. Participants must not initiate symptomatic treatment for MS within 4 weeks prior to enrollment.Exclusion Criteria:Individuals who meet any of the following criteria will be excluded from study entry:■ Pregnant or breastfeeding, or intention of becoming pregnant within the timeframe in which contraception is required:o Participants of childbearing potential must have a negative serum pregnancy test result at Screening Visit 1 and a negative urine pregnancy test within 3 days prior to the initiation of LDC [Day -5]) and prior to P- CD19CD20-ALLO1 administration (on Day 1). If Day -5 is > 14 days from the initial screening serum pregnancy test, the serum pregnancy test must be repeated at the baseline visit. If a urine pregnancy test is positive, it must be confirmed by a serum pregnancy test.■ Participants who have active PML, have had confirmed PML, or for whom the investigator ever had a high degree of clinical suspicion for PML.■ Known or suspected history of HLH / MAS or neurotoxicity with prior therapies.■ Known presence of other neurologic disorders that may mimic MS, including but not limited to, neuromyelitis optica spectrum disease, myelin oligodendrocyte antibody associated disease, Lyme disease, untreated vitamin B-12 deficiency, cerebrovascular or spinal vascular disorders, and untreated hypothyroidism.■ History or known presence of clinically significant brain or spinal cord trauma (e.g., cerebral contusion, spinal cord compression), or clinically significant CNS or spinal cord tumor (e.g., glioma).Attorney Docket No.: 000218-0094-W01■ History of genetically inherited progressive CNS degenerative disorder (e.g., hereditary paraparesis, mitochondrial myopathy, encephalopathy, lactic acidosis, stroke [MELAS] syndrome).■ History or known presence of systemic autoimmune disorders potentially causing neurologic disease aside from MS (e.g., lupus, anti-phospholipid antibody syndrome, Sjogren syndrome, Behget disease, or neurosarcoidosis).■ History of currently active primary or secondary (non-drug-related) immunodeficiency.■ Significant or uncontrolled medical disease which would preclude patient participation.■ High risk for clinically significant bleeding or any condition requiring plasmapheresis, IV Ig, or acute blood product transfusions.■ History of cancer, including solid tumors, hematologic malignancies, and carcinoma in situ, within the past 5 years:o Participants with non-melanomatous carcinomas of the skin that have been treated or excised and have resolved are eligible.■ History or known presence of infectious causes of myelopathy (e.g., syphilis, Lyme disease, human T-lymphotropic virus type 1, herpes zoster myelopathy).■ History of recurrent serious infections or chronic infection.■ Participants with a current diagnosis of epilepsy.■ Exclusions for CSF collection:o Known history of high intracranial pressure.o Local infections at the lumbar puncture site (inclusion is allowed once the infection resolves).o Congenital spine abnormalities or acquired spinal cord abnormalities that prevent a lumbar puncture from being performed safely or comfortably.o Increased risk of bleeding due to low platelet count, uncorrected bleeding diathesis, uncontrolled coagulopathy, or treatment with anticoagulants or antiplatelets (note: anticoagulants and antiplatelets may be temporarily discontinued at the investigator’s discretion as per national or local recommendations on periprocedural antithrombotic management for lumbar puncture [Dodd et al. 2018]).Attorney Docket No.: 000218-0094-W01Clinically significant abnormalities in laboratory test results (including hematology, blood chemistry, and urinalysis), not linked to the participant’s MS disease.■ Presence of severe renal impairment, defined by eGFR < 50 mL / min / 1.73 m2(as estimated using the CKD-EPI 2021 equation) or the need for dialysis or renal transplantation.o Any of the following laboratory parameters:o AST or ALT > 3 x ULNo Amylase or lipase > 2 x ULNo CD4+ T-cell count < 250 / pLo Neutrophils < 1.5 x 103 / pL with one exception:■ Individuals with benign ethnic neutropenia (BEN): ANC < 1300 / pL.■ BEN (also known as constitutional neutropenia) is an inherited cause of mild or moderate neutropenia that is not associated with any increased risk for infections or other clinical manifestations (Atallah-Yunes et al. 2019). BEN is referred to as ethnic neutropenia because of its increased prevalence in people of African descent and other specific ethnic groups.o Hemoglobin < 9 g / dLo Platelet count < 80,000 / pL■ Baseline oxygen saturation < 92% on room air.■ Left ventricular ejection fraction (LVEF) < 45%.■ Known active or latent tuberculosis infection.o If the investigator considers a potential patient to be at an increased risk for infection with Mycobacterium tuberculosis, latent tuberculosis diagnostic procedures must be followed according to local practice standards during the screening period.■ Positive HIV 1 or 2 infection.■ Positive human T-cell lymphotropic virus (HTLV) type 1 or type 2 infection.■ Acute or chronic hepatitis B virus (HB V) (defined as positive hepatitis B surface antigen [HBsAg] serology [Conners et al. 2023]):Attorney Docket No.: 000218-0094-W01o Participants who test negative for HBsAg and hepatitis B surface antibody (HBsAb) tests and positive for total hepatitis B core antibody (HBcAb) test are ineligible.■ Active hepatitis C virus (HCV) antibody test at screening, except in individuals who meet either of the following sets of criteria:o Participants with positive HCV antibody test at screening are eligible only if PCR is negative for HCV RNA. The HCV RNA test will be performed only for patients who have a positive HCV antibody test. ■ Positive Epstein-Barr virus (EBV) viral capsid antigen (VCA) IgM test:o If EBV VCA IgM test is negative, an EBV PCR test should be performed if acute or chronic active EBV infection is clinically suspected. Participants with a PCR-confirmed EBV infection are excluded from the study.■ Positive for human herpes virus (HHV)-6 or HHV-7 infection by PCR.o Participants may be included in the study if they are HHV-6 or HHV-7 IgG antibody positive but PCR negative.■ Any known or suspected active (bacterial, viral, fungal, mycobacterial, or other pathogens) infection (including active SARS-CoV-2 infection) at screening or at the initiation of LDC (Day -5), or any major episode of infection requiring hospitalization or treatment with IV anti-microbials or anti-infective medication within 8 weeks prior to and during screening, or treatment with oral antimicrobials within 2 weeks prior to and during screening.■ Any concomitant disease that may require treatment with systemic corticosteroids or immunosuppressants during the study.■ History of severe allergic reactions attributed to compounds of similar chemical or biologic composition to agents used in study (including, but not limited to, cyclophosphamide and fludarabine used in the LDC, DMSO used as a cryoprotectant in the cell media, etc.).■ Contraindications to mandatory premedications (i.e., acetaminophen and antihistamines) for IRRs or medications used for management of potential risks (corticosteroids), including previous uncontrolled psychosis with corticosteroids or history of closed-angle glaucoma with antihistamines.■ Receipt of any of the following excluded therapies:Attorney Docket No.: 000218-0094-W01Prior treatment with CAR T-cell therapy, gene-therapy product, total body irradiation, bone marrow transplantation, allograft organ transplant, or hematopoietic stem cell transplant at any point.Previous treatment with teriflunomide within 24 months prior to first baseline visit, unless an accelerated elimination with cholestyramine or activated charcoal (if drug was taken < 24 months prior to first baseline visit) has been completed, liver enzymes are in the normal range, and a 4-week washout period prior to the first baseline visit has elapsed. Previous use of cladribine and alemtuzumab, within 12 months of first baseline visit.Previous use of B cell-depleting therapies (e.g., anti-CD20 mAbs, rituximab, ocrelizumab, or ublituximab) within 6 months of first baseline visit (i.e., last infusion must have been at least 6 months before first baseline visit):■ An exception is subcutaneous ofatumumab, which is prohibited with 2 months of first baseline visit (i.e., last infusion must have been at least 2 months before first baseline visit).Previous use of natalizumab within 6 months of first baseline visit. Previous treatment with immunosuppressants including azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, and mitoxantrone within 6 months of first baseline visit.Previous treatment with sphingosine 1-phosphate receptor modulators, including fmgolimod, siponimod, or ozanimod within 14 weeks of first baseline visit.Previous treatment with immunomodulators including INFs-J3 (la or lb), glatiramer acetate, or dimethyl fumarate within 2 weeks of first baseline visit.Systemic corticosteroid therapy > 10 mg / day prednisone equivalent within 30 days of first baseline visit.Treatment with IV Ig or plasmapheresis within 12 weeks of first baseline visit.Inhibitors of Bruton’s tyrosine kinase (BTK), including tolebrutinib or fenebrutinib, within 2 weeks of first baseline visit.Attorney Docket No.: 000218-0094-W01Vaccination with a live or live-attenuated vaccine within 6 weeks of first baseline visit.Vaccination with a killed or inactivated vaccine within 2 weeks of first baseline visit.Treatment with any investigational therapy within 30 days or 5 drugelimination half-lives (whichever is longer) of first baseline visit.Previous treatment with any other immunomodulatory or immunosuppressive medication not already listed above without appropriate washout as described in the applicable local label (washout to be completed prior to first baseline visit):■ If the washout requirements are not described in the applicable local label, then the wash out period must be 5 times the half-life of the medication. The PD effects of the previous medication must also be considered when determining the required time for washout. The Sponsor may be consulted when considering washout periods. Participants screened for this study should not be withdrawn from therapies for the sole purpose of meeting eligibility for the trial.Major surgical procedure within 28 days prior to screening, or anticipation of need for a major surgical procedure during the 12 weeks following P-CD19CD20-ALLO1 infusion.Current alcohol or drug abuse or history of alcohol or drug abuse within 12 months prior to screening or during screening:■ Investigators may elect to obtain a urine drug screen if clinically indicated.Participants with suicidal ideation or behavior within 6 months prior to screening or participants who pose a suicidal risk.Inability to complete an MRI scan (contraindications for MRI, including but not restricted to, pacemaker, cochlear implants, intracranial vascular clips, coronary stent implanted within 8 weeks prior to the time of the intended MRI, etc.), or contraindication to Gd administration.History or presence of clinically significant ECG abnormalities (e.g., PR interval 220 ms), complete left bundle branch block, or second- or third-degree atrioventricular heart block at screening.Attorney Docket No.: 000218-0094-W01o Family history of congenital long QT syndrome or family history of sudden death.o Significant or extensive cardiovascular disease (e.g., cardiac insufficiency, coronary artery disease, cardiomyopathy, congestive heart failure, New York Heart Association Class III or IV or Objective Assessment Class C or D cardiac disease, myocardial infarction within the last 6 months, unstable arrhythmias, or unstable angina) at screening.

[0120] Potential participants will be screened for study eligibility prior to enrollment. Eligible participants will be enrolled before starting a non-myeloablative lymphodepleting chemotherapy (LDC) regimen of cyclophosphamide (300-750 mg / m2 / day) and fludarabine (0-30 mg / m2 / day) administered by IV for 3 days starting on Day -5 (i.e., Days -5, -4, and -3). On Day 1 (3 days after the final day of LDC), participants will receive a single IV infusion of P-CD19CD20-ALLO1 at a starting dose of 60 x 106cells. See Figure 13. Up to four dose levels of P-CD19CD20-ALLO1 will be tested with the maximum dose not exceeding 480 x 106cells. If de-escalation is required, a dose of 20 x 106cells will be administered.

[0121] Participants will be hospitalized for the P-CD19CD20-ALLO1 cell infusion and remain hospitalized for observation for a minimum of 7 days following the day of infusion (Days 1-8). After discharge, participants must remain within an approximately 1-hour travel radius of the treatment site for the remainder of the 28-day period after P-CD19CD20-ALLO1 cell infusion. Participants will undergo long-term follow-up (LTFU) for up to 5 years after P-CD19CD20-ALLO1 cell infusion.

[0122] Participants will be closely monitored for adverse events throughout the study. Adverse events will be graded according to the CTCAE v5.0 (National Cancer Institute's Common Terminology Criteria for Adverse Events, Version 5.0) grading scale. Cytokine release syndrome (CRS), immune effector cell -associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis (HLH) will be assessed according to the American Society for Transplantation and Cellular Therapy (ASTCT) criteria, with specific signs and symptoms associated with the adverse event graded according to CTCAE v5.0. GVHD will be assessed according to the International Bone Marrow Transplant (IBMTR) criteria. Rimiducid may be administered as a rescue therapy at a dose of 0.4 mg / kg and infused over approximatelyAttorney Docket No.: 000218-0094-W012 hours. Blood and CSF samples will be taken at various timepoints during the study to characterize the CK, PD, and immunogenicity of P-CD19CD20-ALLO1.

[0123] Additional cohorts may be added according to the scenarios and criteria listed in Table 3 below. If the frequency or severity of adverse events suggests that the MTD has been exceeded, dosing will be halted or modified, as deemed appropriate. Table 3: Scenarios for Optional CohortsCK = cellular kinetic; ddPCR= droplet digital polymerase chain reaction; LDC =lymphodepleting chemotherapy; PD = pharmacodynamic; PMS = progressive multiple sclerosis;RMS = relapsing multiple sclerosis; SMC= Safety Monitoring Committee.

[0124] Participants will be evaluated for an MS relapse throughout the study. EDSS, nine-hole peg test (9-HPT), timed 25-foot walk test (T25FWT), and symbol digit modalities test (SDMT) should be performed within 7 days from the onset of the new or worsening neurologic event to establish the current neurologic status of the patient.

[0125] The primary objective for this study is to evaluate the safety and tolerability of P-CD19CD20-ALLO1 on the basis of the following endpoints:■ Nature and incidence of dose-limiting toxicity (DLT) at each dose level of P-CD19CD20-ALLO1;■ Incidence and severity of adverse events, with severity determined according to the CTCAE v5.0 grading scale, except in cases of cytokine release syndromeAttorney Docket No.: 000218-0094-W01(CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis (HLH), which will be assessed according to American Society for Transplantation and Cellular Therapy (ASTCT) criteria, and graft-versus-host disease (GVHD), which will be assessed according to International Bone Marrow Transplant Registry (IBMTR) criteria;■ Incidence and severity of acute MRI activity (On Day 15 and Day 29 MRI scan);■ Incidence of clinically significant abnormal vital signs;■ Incidence of abnormal clinical laboratory test results; and■ Incidence of abnormal ECG parameters.

[0126] The secondary objectives for this study are:■ To characterize the cellular kinetic (CK) profile of P-CD19CD20-ALLO1 on the basis of the number of CAR transgene copies in the blood at specified timepoints by droplet digital (ddPCR);■ To evaluate the pharmacodynamic (PD) effects of P-CD19CD20-ALLO1 on circulating B cells on the basis of B-cell levels in the blood (CD19+, absolute counts in blood) at specified timepoints; and■ To evaluate the immunogenicity of P-CD19CD20-ALLO1 on the basis of achieving humoral immunity: Prevalence of anti-CAR-T antibodies at baseline and their incidence during the study.

[0127] The exploratory endpoints for this study are listed in Table 4 below:Attorney Docket No.: 000218-0094-W01Table 4: Exploratory Objectives and Corresponding Endpoints12w-CDP = 12-week confirmed disability progression; 24w-cCDP = composite 24-week confirmed disability progression; 9-HPT = nine-hole peg test; ADA = anti-drug antibody; ARR= annualized relapse rate; BCR = B-cell receptor; CK = cellular kinetics; CRS = cytokine release syndrome; CSF= cerebrospinal fluid; CTCAE v5.0 = National Cancer Institute's Common Terminology Criteria for Adverse Events, Version 5.0; GFAP = glial fibrillary acidic protein; IL = interleukin; LDC = lymphodepleting chemotherapy; MRI = magnetic resonance imaging; MS = multiple sclerosis; NfL= neurofilament light chain; PD = pharmacodynamic; PK = pharmacokinetic; PBMC =Attorney Docket No.: 000218-0094-W01peripheral blood mononuclear cell; SDMT= symbol digit modalities test; T25FWT = timed 25-foot walk test.

[0128] Clinical disease activity assessments that will be used in this study include the Expanded Disability Status Scale (EDSS), the Nine-Hole Peg Test (9-HPT), the Timed 25-Foot Walk Test (T25FWT), and the Symbol Digit Modalities Test (SDMT).Expanded Disability Status Scale (EDSS)

[0129] The EDSS is the most commonly used clinician-reported outcome (ClinRO) measure for quantifying changes in the disability level of patients with MS over time. The EDSS is based on a standard neurologic examination, incorporating functional systems (visual, brainstem, pyramidal, cerebellar, sensory, bowel and bladder, and cerebral or mental) that are rated and then scored as a Functional Systems Score (FSS), and ambulation, which is scored as ambulation score. Each FSS is an ordinal clinical rating scale ranging from 0 to 5 or 6 and an ambulation score that is rated from 0 to 16. These ratings are then used in conjunction with observations, as well as information, concerning ambulation and use of assistive devices to determine the total EDSS score. The EDSS is a disability scale that ranges in 0.5-point steps from 0 (normal) to 10.0 (death) (Kurtzke 1983; Kappos 2011). Note that the following items do not need to be scored for this study: sexual dysfunction and fatigue.

[0130] At screening, the EDSS will be used to assist in eligibility determination. Participants with PMS must have an EDSS score from 3 to 6 inclusive at screening and documented EDSS progression of > 1 point in the 2 years prior to screening (or a written summary of the clinical evidence of disability progression in the 2 years prior to screening) to be considered eligible for enrollment. For participants with RMS, an EDSS score from 0 to 6 inclusive at screening is required to be considered eligible for enrollment.Nine-Hole Peg Test (9-HPT)

[0131] The 9-HPT is a performance measure used to assess upper extremity (arm and hand) function (Goodkin et al. 1988; Fischer et al. 1999). The test consists of a container with nine pegs and a wood or plastic block with nine empty holes. The patient has to pick up each of the nine pegs one at a time and, as quickly as possible, place them in the nine holes. Once all the pegs are in the holes, the patient is to remove them again one at a time as quickly as possible and replace them into the container.Attorney Docket No.: 000218-0094-W01The total time to complete the task is recorded. Both the dominant and non-dominant hands are tested twice (two consecutive trials of the dominant hand, followed immediately by two consecutive trials of the non-dominant hand).

[0132] Detailed guidance for the administration of the 9-HPT is described in the MSFC Administration and Scoring Manual (Fischer et al. 2001). A 20% change from baseline is typically considered clinically meaningful (Feys et. al. 2017).Timed 25-Foot Walk Test (T25FWT)

[0133] The T25FWT test is a performance measure used to assess walking speed based on a timed 25-foot walk. The patient is directed to start at one end of a clearly marked 25-foot course and is instructed to walk 25 feet as quickly and safely as possible. The investigator will time the patient from the start of the walk to the end of the 25 feet. The task is immediately administered again by having the patient walk back the same distance. The score for the T25FWT is the average of the two completed trials. Patients may use assistive devices (e.g., cane, crutch, or rollator) when performing the task. The investigator should assess which assistive device is necessary and use the same assistive device at each study visit, whenever possible. Non-wheeled walkers should not be used. Circumstances that may affect the patient’s performance, or if the patient cannot complete the T25FWT twice, should be reported.

[0134] The T25FWT will be administered as described in the MSFC Administration and Scoring Manual (Fischer et al. 2001). A 20% change from baseline of the averaged T25FWT is typically considered clinically meaningful (Hobart et. al.2013; EMA 2019).Symbol Digit Modalities Test (SDMT)

[0135] The SDMT is a performance measure that has demonstrated sensitivity in detecting not only the presence of cognitive impairment but also changes in cognitive functioning over time and in response to treatment (Smith 1982). The SDMT is recognized as being particularly sensitive to slowed processing of information that is commonly seen in MS (Benedict et al. 2017). The SDMT is brief, is easy to administer, and involves a simple substitution task that normal children and adults can easily perform. Using a reference key, the examinee has 90 seconds to pair specific numbers with given geometric figures. Responses will be collected orally. A four-point change from baseline is typically considered clinically meaningful (Benedict et. al. 2017).Attorney Docket No.: 000218-0094-W01Magnetic Resonance Imaging

[0136] MRI scans will be used to monitor volumetric and lesion changes in the CNS and potentially other pathophysiology, such as inflammation and neurodegeneration. Screening MRI scans will be obtained and checked for quality, only if needed to establish eligibility. Results from the screening MRI should be assessed before CSF sampling to avoid exposing participants to an invasive procedure in case of exclusionary findings.

[0137] All MRI scans will be reviewed and evaluated by a qualified, certified expert.American Society for Transplantation and Cellular Therapy (ASTCT) Consensus Grading

[0138] The severity of CRS will be assessed through use of the ASTCT grading scale for CRS (see Table 5, infra).Table 5: ASTCT Grading Scale for Cytokine Release Syndrome> > > >ASTCT = American Society for Transplantation and Cellular Therapy; BiPAP= bi-level positive airway pressure; CPAP= continuous positive airway pressure; CRS = cytokine release syndrome; CTCAE v5.0D National Cancer Institute's Common Terminology Criteria for Adverse Events, Version 5.0.Note: Organ toxicities associated with CRS may be graded according to CTCAE v5.0, but they do not influence CRS grading.a Fever may not be prominent in participants who have received antipyretics, corticosteroids, or anti-cytokine therapy. In such cases, the grade is driven by hypotension and / or hypoxia.bLow flow is defined as oxygen delivered at < 6 L / min via nasal cannula or oxygen delivered via blow-by. High flow is defined as oxygen delivered at > 6 L / min.Attorney Docket No.: 000218-0094-W01cParticipant may or may not be receiving vasopressin but must be receiving one vasopressor other than vasopressin.dParticipant may or may not be receiving vasopressin but must be receiving two or more vasopressors other than vasopressin.

[0139] The severity of ICANS will be assessed through use of the ASTCT grading scale for ICANS (see Table 6, infra), which takes into account the ICE score (see Table 7, infra).Table 6: ASTCT Grading Scale for Immune Effector Cell-Associated Neurotoxicity Syndrome>ASTCT = American Society for Transplantation and Cellular Therapy; CTCAE v5.0 = National Cancer Institute's Common Terminology Criteria for Adverse Events, Version 5.0; ICANS = immune effector cell- associated neurotoxicity syndrome; ICE = immune effector cell-associated encephalopathy; NA=not applicable.aICANS grade is determined by the most severe event (ICE score, or neurologic symptoms of depressed level of consciousness, seizure activity, motor weakness, or elevated intracranial pressure or cerebral edema) not attributable to any other cause. For example, a participant with an ICE score of 3 who has a generalized seizure is classified as Grade 3 ICANS.bA participant with an ICE score of 0 who is awake with global aphasia may be classified as Grade 3 ICANS, but a participant with an ICE score of 0 who is unarousable may be classified as Grade 4 ICANS.cDepressed level of consciousness should not be attributable to any other cause (e.g., sedating medication).dTremors and myoclonus associated with immune effector cell therapies do not influence ICANS grading. They may be graded according to CTCAE v5.0.eIntracranial hemorrhage with or without associated edema is not considered a neurotoxicity feature and does not influence ICANS grading. It may be graded according to CTCAE v5.0.Table 7: ICE ScoreAttorney Docket No.: 000218-0094-W01" " """"ICE = immune effector cell-associated encephalopathy.a1 point for each correct answer.

[0140] The severity of HLH will be assessed through use of the ASTCT grading scale for HLH (see Table 8, infra).Table 8: ASTCT Grading Scale for Hemophagocytic LymphohistiocytosisASTCT = American Society for Transplantation and Cellular Therapy; CRS = cytokine release syndrome; CTCAE v5.0= National Cancer Institute's Common Terminology Criteria for Adverse Events, Version 5.0; HLH= hemophagocytic lymphohistiocytosis; ULN= upper limit of normal.Note: The ASTCT grading scale (Hines et al. 2023) is based on the CTCAE v5.0 "immune system disorders, other" category.aHLH is a pathological and biochemical hyperinflammatory syndrome that may present with fever, hepatosplenomegaly, organ failure, and neurologic toxicities and is associated with progression or new onset of hyperferritinemia, cytopenias, coagulopathy with hypofibrinogenemia, and / or elevation of liver enzymes. HLH and CRS that occur subsequent to T cell-redirecting therapy have overlapping features, and HLH-like symptoms may be seen in individuals with severe CRS. Although HLH is distinct from CRS in this setting, overlapping clinical features may make it difficult to distinguish HLH from CRS. Temporally, HLH may have a delayed onset and can occur as CRS is resolving or after CRS has resolved. In addition, HLH can occur in the absence of CRS.International Bone Marrow Transplant Registry (IBMTR) Grading Scale For Graft Versus Host Disease.

[0141] The severity of GVHD will be assessed through use of the IBMTR grading scale for GVHD (see Table 9, infra).Attorney Docket No.: 000218-0094-W01Table 9: Criteria for IBMTR Severity Index for Acute GVHD < < <> >>BSA = body surface area; IBMTR= International Bone Marrow Transplant Registry.aAssign index based on maximum involvement in an individual organ system. Source: Rowlings et al. 1997; Schoemans et al. 2018.

Claims

Attorney Docket No.: 000218-0094-W01CLAIMS1. A method of treating multiple sclerosis (MS) in a subject in need thereof comprising administering a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (C AR-T cell) to the subj ect.

2. Use of a P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) in the manufacture of a medicament for treating multiple sclerosis (MS) in a subject in need thereof.

3. A P-CD19CD20-ALLO1 chimeric antigen receptor T cell (CAR-T cell) for use in treating multiple sclerosis (MS) in a subject in need thereof.

4. The method according to claim 1, the use according to claim 2, or the CAR-T cell for use according to claim 3, wherein the MS is progressive MS (PMS).

5. The method, use or CAR-T for use according to claim 4, wherein the PMS is primary PMS (PPMS) or non-active secondary PMS (naSPMS).

6. The method according to claim 1, the use according to claim 2, or the CAR-T cell for use according to claim 3, wherein the MS is relapsing MS (RMS).

7. The method, use or CAR-T for use according to claim 6, wherein the RMS is relapsing remitting MS (RRMS) or active secondary progressive MS (aSPMS).

8. The method, use or CAR-T cell for use according to any one of claims 4-7, wherein the MS is diagnosed according to the revised 2017 McDonald Criteria.

9. The method according to any one of claims 1 and 4-8, the use according to any one of claims 2 and 4-8, or the CAR-T cell for use according to any one of claims 3-8, wherein the CAR-T cell is administered to the subject intravenously or is formulated to be administered to the subject intravenously.

10. The method according to any one of claims 1 and 4-9, the use according to any one of claims 2 and 4-9, or the CAR-T cell for use according to any one of claims 3-9, wherein the CAR-T cell is administered as a single dose or is formulated to be administered as a single dose.

11. The method according to any one of claims 1 and 4-9, the use according to any one of claims 2 and 4-9, or the CAR-T cell for use according to any one of claims 3-9, wherein the CAR-T cell is administered in multiple doses or is formulated to beAttorney Docket No.: 000218-0094-W01administered in multiple doses, optionally wherein each of the multiple doses is administered between 3 and 5 years after the previous dose.

12. The method according to any one of claims 1 and 4-11, the use according to any one of claims 2 and 4-11, or the CAR-T cell for use according to any one of claims 3- 11, wherein the CAR-T cell is administered at a dose of about 5 x 106cells to about 1.2 x 109cells or is formulated to be administered at a dose of about 5 x 106to about 1.2 x 109cells.

13. The method according to any one of claims 1 and 4-12, the use according to any one of claims 2 and 4-12, or the CAR-T cell for use according to any one of claims 3- 12, wherein administration of the CAR-T cell results in (a) a detectable number of CAR transgene copies in the blood of the subject, (b) a detectable number of CAR-T cells in the blood of the subject, (c) the depletion of B-cells in the blood or cerebrospinal fluid (CSF) of the subject by more than 90 percent as compared to prior to treatment, (d) a reduction in the number of gadolinium lesion counts and no new or enlarging T2 lesion counts identified by magnetic resonance imaging (MRI) as compared to prior to treatment, (e) no disability progression as compared to prior to treatment measured by the confirmed composite disability progression (cCDP) score confirmed at 12 or 24 weeks (24w-cCDP or 12w-cCDP) after treatment, or (f) an improvement in MS-associated biomarkers (e.g., oligoclonal bands, NfL, and GFAP) as compared to prior to treatment.

14. The method according to any one of claims 1 and 4-13, the use according to any one of claims 2 and 4-13, or the CAR-T cell for use according to any one of claims 3- 13, wherein administration of the CAR-T cell results in (a) an incidence of Grade 2 cytokine release syndrome (CRS) less than 30%, (b) an incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) less than 20%, (c) an incidence of hemophagocytic lymphohistiocytosis (HLH) less than 10%, (d) a risk of serious infection no more than 10% more compared to the incidence of infection resulting from standard of care 6 months after administration of the CAR-T cell, (e) an incidence of immune rejection and graft-versus-host disease (GVHD) less than 10%, (f) an incidence of progressive multifocal leukoencephalopathy (PML) less than 10%, (g) an incidence of CAR T cell-driven worsening of MS in less than 20% of subjects administered the CAR-T cell, or (h) an incidence of new T-cell malignancies less than 0.1%.Attorney Docket No.: 000218-0094-W0115. The method according to any one of claims 1 and 4-14, the use according to any one of claims 2 and 4-14, or the CAR-T cell for use according to any one of claims 3-14, wherein the subject has not been previously administered a therapeutic agent to treat MS.

16. The method according to any one of claims 1 and 4-14, the use according to any one of claims 2 and 4-14, or the CAR-T cell for use according to any one of claims 3-14, wherein the subject has been previously administered one or more therapeutic agents to treat MS.

17. The method, use, or CAR-T cell for use according to claim 16, wherein the subject is refractory to the one or more previously administered therapeutic agents.

18. The method, use, or CAR-T cell for use according to claim 16 or 17, wherein the one or more previously administered therapeutic agents are selected from an immunomodulator, a cell migration modulator, a lymphocyte depleting therapy, a kinase inhibitor, or a corticosteroid.

19. The method, use, or CAR-T cell for use according to claim 18, wherein the immunomodulator is selected from the group consisting of interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, teriflunomide, and mitoxantrone.

20. The method, use, or CAR-T cell for use according to claim 18, wherein the cell migration modulator is selected from the group consisting of fmgolimod, siponimod, ozanimod, and natalizumab.

21. The method, use, or CAR-T cell for use according to claim 18, wherein the lymphocyte depleting therapy is selected from the group consisting of alemtuzumab, cladribine, ocrelizumab, rituximab, ublituximab, and ofatumumab.

22. The method, use, or CAR-T cell for use according to claim 18, wherein the kinase inhibitor is selected from the group consisting of baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, and fenebrutinib.

23. The method, use, or CAR-T cell for use according to claim 18, wherein the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.Attorney Docket No.: 000218-0094-W0124. The method according to any one of claims 1 and 4-23, the use according to any one of claims 2 and 4-23, or the CAR-T cell for use according to any one of claims 3-23, wherein the CAR-T cell is administered or formulated to be administered in combination with one or more additional therapeutic agents.

25. The method, use, or CAR-T cell for use according to claim 24, wherein the CAR-T cell is administered or formulated to be administered sequentially with the one or more additional therapeutic agents.

26. The method, use, or CAR-T cell for use according to claim 24 or 25, wherein the one or more additional therapeutic agents is selected from an immunosuppressant, a corticosteroid, a kinase inhibitor, or a cytokine blocker.

27. The method, use, or CAR-T cell for use according to claim 26, wherein the immunosuppressant is selected from the group consisting of cyclophosphamide, tocilizumab, siltuximab and anakinra.

28. The method, use, or CAR-T cell for use according to claim 26, wherein the corticosteroid is selected from the group consisting of prednisone, dexamethasone, and methylprednisolone.

29. The method, use, or CAR-T cell for use according to claim 26, wherein the kinase inhibitor is ruxolitinib.

30. The method, use, or CAR-T cell for use according to claim 26, wherein the cytokine blocker is selected from the group consisting of ruxolitinib, etoposide, or emapalumab.

31. The method according to any one of claims 1 and 4-30, the use according to any one of claims 2 and 4-30, or the CAR-T cell for use according to any one of claims 3-30, wherein the subject receives a lymphodepleting chemotherapy (LDC) regimen prior to the administration of CAR-T cell.

32. The method, use, or CAR-T cell for use according to claim 31, wherein the LDC regimen comprises cyclophosphamide (300-750 mg / m2IV) and fludarabine (0-30 mg / m2IV) for 3 consecutive days.

33. The method, use, or CAR-T cell for use according to claim 31 or 32, wherein the LDC regimen is administered on Days -5, -4, and -3, and the CAR-T cells is administered or to be administered on Day 1.

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