Combination immunosuppression for inhibiting an immune response and enabling immunogen administration and re-administration

Abstract

The present disclosure provides compositions and methods for inhibiting or preventing an immune response to an immunogen (e.g., an immunogenic delivery vehicle) in a subject in need thereof, comprising administering to the subject an effective amount of a plasma cell depleting agent, e.g., an antigen-binding molecule that binds to B cell maturation antigen (BCMA) and cluster of differentiation 3 (CD3) (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof) or a B cell depleting agent, e.g., an antigen-binding molecule that binds to CD20 and CD3 (e.g., an anti-CD20xCD3 bispecific antibody, or a functional fragment thereof), either alone or in combination with one another, and / or in combination with an immunoglobulin depleting agent such as a neonatal fragment crystallizable (Fc) receptor (FcRn) blocker (e.g., efgartigimod).

Description

CROSS-REFERENCE TO RELATED APPLICATION

[001] This patent application claims the benefit of U.S. Provisional Application No. 63 / 625,524, filed January 26, 2024, the disclosure of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 16, 2025, is named 250298_000776_SL.xml and is 54,578 bytes in size. FIELD OF THE INVENTION

[003] The present disclosure provides compositions and methods for inhibiting or preventing an immune response to an immunogen (e.g., an immunogenic delivery vehicle) in a subject in need thereof, comprising administering to the subject an effective amount of a plasma cell depleting agent, e.g., an antigen-binding molecule that binds to B cell maturation antigen (BCMA) and CD3 (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof), either alone or in combination with a B cell depleting agent (e.g., anti-CD20xCD3 bispecific antibody, or a functional fragment thereof), and / or an immunoglobulin depleting agent such as a neonatal fragment crystallizable (Fc) receptor (FcRn) blocker (e.g., efgartigimod). Further disclosed herein are methods and compositions for inhibiting an immune response to an immunogen in a subject in need thereof by using a B cell depleting agent administered alone. BACKGROUND

[004] Adeno-associated virus (AAV)-based vectors hold tremendous promise to transform treatment of genetic diseases. Yet, the potential of AAV gene therapy has so far been limited by development of host antibodies (e.g., neutralizing antibodies (nAbs)) that block transduction or affect uptake on subsequent exposures. Clinically, the inability to re-dose AAVs presents challenges because efficacy cannot be restored if transgene expression is subtherapeutic or lost (e.g., due to cell division, silencing, or a cytotoxic immune response). Moreover, due to natural AAV exposure, many patients develop nAbs prior to treatment that render them ineligible for even a single dose. Therefore, strategies that prevent or attenuate anti-AAV nAb responses could vastly expand the utility and accessibility of existing AAV gene therapies, while safeguarding eligibility for future AAV-based advances. SUMMARY

[005] As specified in the Background section above, there exists a need in the art to enhance the efficacy of treatments with, e.g., recombinant vectors (e.g., AAV). This can be achieved, e.g., by inhibiting or preventing an immune response against such recombinant vectors and / or their transgene products (e.g., therapeutic polypeptides or polynucleotides encoded by the transgene), thereby improving efficacy and reducing toxicity of gene therapy. Such advancements would allow for stepwise dosing and / or effective re-administration (i.e., redosing) of the recombinant vectors (e.g., AAV) to increase or maintain the level of a transgene expression. The present disclosure addresses these and other needs.

[006] In one aspect, provided herein is a method for inhibiting or preventing an immune response to an immunogen in a subject in need thereof, wherein the subject has pre-existing immunity against the immunogen, the method comprising administering to the subject an effective amount of a plasma cell depleting agent.

[007] In another aspect, provided herein is a method for inhibiting or preventing generation of antibodies to an immunogen in a subject in need thereof, wherein the subject has pre-existing immunity against the immunogen, the method comprising administering to the subject an effective amount of a plasma cell depleting agent.

[008] In another aspect, provided herein is a method for increasing effectiveness of readministration of an immunogen to a subject in need thereof, wherein the subject has preexisting immunity against the immunogen, the method comprising administering to the subject an effective amount of a plasma cell depleting agent.

[009] In some embodiments, the immunogen re-administration occurs via the same administration route as its prior administration.

[010] In some embodiments, the immunogen re-administration occurs via a different administration route than its prior administration.

[011] In some embodiments, the method comprises determining the presence of neutralizing antibodies to the immunogen in the subject.

[012] In some embodiments, the plasma cell depleting agent is administered before the administration of the immunogen.

[013] In some embodiments, the plasma cell depleting agent is administered simultaneously with the administration of the immunogen.

[014] In some embodiments, the plasma cell depleting agent is administered after the administration of the immunogen.

[015] In some embodiments, the immunogen is administered two or more times and the plasma cell depleting agent is administered before and / or between each of the administrations of the immunogen.

[016] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.

[017] In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

[018] In another aspect, provided herein is a method for increasing or maintaining the level of a transgene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of a plasma cell depleting agent.

[019] In some embodiments, the method comprises determining the presence of neutralizing antibodies to the immunogen in the subject.

[020] In some embodiments, the transgene is delivered to the subject via an immunogenic delivery vehicle.

[021] In some embodiments, the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene.

[022] In some embodiments, the level of transgene expression is increased or maintained by inhibiting antibody responses to the polypeptide or polynucleotide encoded by the transgene.

[023] In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, a protozoal vector, or a mammalian cell.

[024] In some embodiments, the immunogenic delivery vehicle is a viral vector.

[025] In another aspect, provided herein is a method for increasing effectiveness of administration of a subsequently administered viral vector following administration of an originally administered viral vector in a subject in need thereof, the method comprising administering to the subject an effective amount of a plasma cell depleting agent, wherein the subsequently administered viral vector is of the same or similar viral origin as the originally administered viral vector.

[026] In some embodiments, the method comprises determining the presence of neutralizing antibodies to the immunogen in the subject.

[027] In some embodiments, the subsequently administered viral vector is administered via the same administration route as the originally administered viral vector.

[028] In some embodiments, the subsequently administered viral vector is administered via a different administration route from the originally administered viral vector.

[029] In some embodiments, the plasma cell depleting agent is administered before the administration of the subsequently administered viral vector(s).

[030] In some embodiments, the plasma cell depleting agent is administered simultaneously with the administration of the subsequently administered viral vector(s).

[031] In some embodiments, the subsequently administered viral vectors are administered two or more times and the plasma cell depleting agent is administered before and / or between each of the administrations of the subsequently administered viral vectors.

[032] In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, a retrovirus, or an oncolytic virus.

[033] In some embodiments, the viral vector is AAV.

[034] In some embodiments, the plasma cell depleting agent is capable of depleting long- lived plasma cells (LLPC).

[035] In some embodiments, the plasma cell depleting agent is a B cell maturation antigen (BCMA) targeting agent.

[036] In some embodiments, the BCMA targeting agent is a chimeric antigen receptor (CAR) against BCMA or an anti-BCMA antibody or a functional fragment thereof.

[037] In some embodiments, the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent.

[038] In some embodiments, the anti-BCMA antibody is a multispecific antibody or a functional fragment thereof.

[039] In some embodiments, the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3.

[040] In some embodiments, the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof.

[041] In some embodiments, the anti-BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

[042] In some embodiments, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18.

[043] In some embodiments, the first antigen-binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[044] In some embodiments, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18.

[045] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[046] In some embodiments, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively.

[047] In some embodiments, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively.

[048] In some embodiments, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

[049] In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1.

[050] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

[051] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[052] In some embodiments, the method further comprises administering to the subject an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent.

[053] In some embodiments, the B cell depleting agent is administered before, at the same time as, or after the plasma cell depleting agent.

[054] In some embodiments, the immunoglobulin depleting agent is administered after the plasma cell depleting agent.

[055] In some embodiments, the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

[056] In some embodiments, the B cell depleting agent is an agent that binds to a B cell surface molecule.

[057] In some embodiments, the B cell depleting agent is selected from anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD79 antibodies, multispecific antibodies combining two or more of any of said antibody specificities, multispecific antibodies combining any of said antibody specificities with anti-CD3 antibodies, functional fragments of any of said antibodies, and any combinations thereof.

[058] In some embodiments, the B cell depleting agent comprises an anti-CD20 antibody or a functional fragment thereof and an anti-CD19 antibody or a functional fragment thereof.

[059] In some embodiments, the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof.

[060] In some embodiments, the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof.

[061] In some embodiments, the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3.

[062] In some embodiments, the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

[063] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[064] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[065] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[066] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[067] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[068] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

[069] In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1.

[070] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

[071] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[072] In some embodiments, the B cell depleting agent is an agent targeting a B cell survival factor.

[073] In some embodiments, the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof.

[074] In some embodiments, the immunoglobulin depleting agent is capable of accelerating IgG clearance.

[075] In some embodiments, the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker.

[076] In some embodiments, the FcRn blocker is selected from Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), Nipocalimab (M281), Orilanolimab (SYNT001), IM\ / T-1402, and any combinations thereof.

[077] In some embodiments, the method further comprises plasmapheresis, therapeutic plasma exchange, or immunoadsorption.

[078] In a further aspect, provided herein is a pharmaceutical composition comprising (i) a plasma cell depleting agent, (ii) a B cell depleting agent and / or an immunoglobulin depleting agent, and (iii) a pharmaceutically acceptable carrier and / or excipient.

[079] In a further aspect, provided herein is a pharmaceutical composition comprising (i) an immunogen, (ii) a plasma cell depleting agent, (iii) optionally, a B cell depleting agent and / or an immunoglobulin depleting agent, and (iv) a pharmaceutically acceptable carrier and / or excipient.

[080] In another aspect, provided herein is a kit comprising (i) a plasma cell depleting agent, (ii) a B cell depleting agent and / or an immunoglobulin depleting agent, and (iii) optionally, instructions for use.

[081] In another aspect, provided herein is a kit comprising (i) an immunogen, (ii) a plasma cell depleting agent, (iii) optionally a B cell depleting agent and / or an immunoglobulin depleting agent, and (iv) optionally, instructions for use.

[082] In a further aspect, provided herein is a method for inhibiting or preventing an immune response to an immunogen in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof.

[083] In some embodiments, inhibiting the immune response comprises suppression of numbers and frequencies of immunogen-specific B cells.

[084] In some embodiments, inhibiting the immune response comprises suppression of immunogen-specific IgG and / or IgM responses.

[085] In a further aspect, provided herein is a method for inhibiting or preventing generation of neutralizing antibodies to an immunogen in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof.

[086] In a further aspect, provided herein is a method for increasing effectiveness of readministration of an immunogen to a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof.

[087] In some embodiments, the immunogen re-administration occurs via the same administration route as its prior administration.

[088] In some embodiments, the immunogen re-administration occurs via a different administration route than its prior administration.

[089] In some embodiments, the subject does not have a pre-existing immunity against the immunogen.

[090] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogen to the subject.

[091] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogen to the subject.

[092] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogen to the subject.

[093] In some embodiments, the immunogen is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogen.

[094] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.

[095] In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

[096] In another aspect, provided herein is a method for increasing or maintaining the level of a transgene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof.

[097] In some embodiments, the transgene is delivered to the subject via an immunogenic delivery vehicle.

[098] In some embodiments, the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene.

[099] In some embodiments, the level of transgene expression is increased or maintained by inhibiting antibody responses to a polypeptide or polynucleotide encoded by the transgene.

[0100] In some embodiments, the subject does not have a pre-existing immunity against the immunogenic delivery vehicle and / or a polypeptide or polynucleotide encoded by the transgene.

[0101] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogenic delivery vehicle to the subject.

[0102] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogenic delivery vehicle to the subject.

[0103] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogenic delivery vehicle to the subject.

[0104] In some embodiments, the immunogenic delivery vehicle is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogenic delivery vehicle.

[0105] In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.

[0106] In some embodiments, the immunogenic delivery vehicle is a viral vector.

[0107] In a further aspect, provided herein is a method for increasing effectiveness of a subsequently administered viral vector following an originally administered viral vector in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof, wherein the subsequently administered viral vector is of the same or similar viral origin as the originally administered viral vector.

[0108] In some embodiments, the subsequently administered viral vector is administered via the same administration route as the originally administered viral vector.

[0109] In some embodiments, the subsequently administered viral vector is administered via a different administration route from the originally administered viral vector.

[0110] In some embodiments, the subject does not have a pre-existing immunity against the viral vectors.

[0111] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the originally administered viral vector to the subject.

[0112] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the originally administered viral vector and / or subsequently administered viral vector to the subject.

[0113] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the originally administered viral vector but before administering the subsequently administered viral vector to the subject.

[0114] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the subsequently administered viral vector to the subject.

[0115] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the viral vectors to the subject.

[0116] In some embodiments, the viral vectors are derived from an adeno-associated virus (AAV), an adenovirus, or a retrovirus.

[0117] In some embodiments, the viral vectors are derived from AAV.

[0118] In some embodiments, the subsequently administered AAV vector has a capsid derived from the same AAV serotype as the originally administered AAV vector.

[0119] In some embodiments, the retrovirus is a lentivirus.

[0120] In some embodiments, the viral vectors are derived from an oncolytic virus.

[0121] In some embodiments, the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.

[0122] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0123] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0124] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0125] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0126] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0127] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

[0128] In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1.

[0129] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

[0130] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[0131] In another aspect, provided herein is a composition comprising an immunogen and an anti-CD20xCD3 bispecific antibody or a functional fragment thereof and optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

[0132] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.

[0133] In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

[0134] In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.

[0135] In some embodiments, the immunogenic delivery vehicle is a viral vector.

[0136] In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, or a retrovirus.

[0137] In some embodiments, the viral vector is derived from AAV.

[0138] In some embodiments, the retrovirus is a lentivirus.

[0139] In some embodiments, the viral vector is derived from an oncolytic virus.

[0140] In some embodiments, the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.

[0141] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0142] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0143] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0144] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0145] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0146] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

[0147] In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1.

[0148] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

[0149] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[0150] In another aspect, provided herein is a kit comprising (i) an immunogen, (ii) an anti-CD20xCD3 bispecific antibody or a functional fragment thereof, and (iii) optionally, instructions for use.

[0151] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.

[0152] In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

[0153] In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.

[0154] In some embodiments, the immunogenic delivery vehicle is a viral vector.

[0155] In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, or a retrovirus.

[0156] In some embodiments, the viral vector is derived from AAV.

[0157] In some embodiments, the retrovirus is a lentivirus.

[0158] In some embodiments, the viral vector is derived from an oncolytic virus.

[0159] In some embodiments, the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.

[0160] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0161] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0162] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0163] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0164] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0165] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

[0166] In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1.

[0167] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

[0168] In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[0169] These and other aspects described herein will be apparent to those of ordinary skill in the art in the following description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1 shows an experimental timeline for the study described in Examples 1,2, and 3.

[0171] Figure 2 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 and anti-CD20 antibodies (anti-CD19 / CD20 antibodies), or combination thereof, on anti-AAV8 capsid IgG titers over time in mice previously treated with recombinant AAV8 vector.

[0172] Figure 3 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second AAV8 vector in mice previously treated with recombinant AAV8 vector, as measured by Taqman quantitative real-time polymerase chain reaction (PCR) of green fluorescent protein (GFP) transgene DNA.

[0173] Figure 4 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second recombinant AAV8 vector in mice previously treated with a first recombinant AAV8 vector, as measured by Taqman quantitative real-time reverse-transcription PCR of GFP transgene RNA.

[0174] Figures 5A-5B show the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second recombinant AAV8 vector in mice previously treated with a first recombinant AAV8 vector, as measured by GFP immunohistochemical (IHC) staining of formalin-fixed paraffin embedded liver sections. Figure 5A shows GFP-positive area quantified using HALO software (Indica labs). Figure 5B shows representative images.

[0175] Figures 6A-6J show flow cytometry analysis of B cell and plasma cell frequencies and counts in bone marrow and spleen following treatment with anti-BCMAxCD3 bispecific antibody, FcRn blockade, anti-CD19 / CD20 antibodies, or combinations thereof. Figure 6A shows bone marrow plasma cell frequencies. Figure 6B shows spleen plasma cell frequencies. Figure 6C shows spleen naive B cell frequencies. Figure 6D shows spleen total memory B cell frequencies. Figure 6E shows spleen AAV-specific memory B cell frequencies. Figure 6F shows bone marrow plasma cell counts. Figure 6G shows spleen plasma cell counts. Figure 6H shows spleen naive B cell counts. Figure 6I shows spleen total memory B cell counts. Figure 6J shows spleen AAV-specific memory B cell counts.

[0176] Figure 7 shows the effect of efgartigimod on serum drug concentration of REGN5458 (BCMAxCD3).

[0177] Figure 8 shows an experimental timeline for the study described in Example 10.

[0178] Figures 9A-9B show the effect of plasma cell depletion, B cell depletion, neonatal Fc receptor blockade, and combinations thereof, on naturally-occurring anti-AAV antibody titers in cynomolgus macaques. AAV8 neutralizing antibody (NAb) titer levels are presented for each treatment group over the duration of the study (Figure 9A) and specifically at Study Day 29 (Figure 9B).

[0179] Figure 10 shows an experimental timeline for the study described in Examples 11 and 12.

[0180] Figures 11A-11C show a comparison of the effect of CD20xCD3-mediated versus anti-CD20-mediated B cell depletion on the development of anti-AAV IgM antibody titers (Figure 11 A) and anti-AAV IgG antibody titers (Figures 11B-11C) in mice.

[0181] Figures 12A-12C show a comparison of the effect of CD20xCD3-mediated versus anti-CD20-mediated B cell depletion on AAV transduction (Figure 12A) and transgene expression (Figures 12B-12C) following vector re-administration in mice.

[0182] Figure 13 shows an experimental timeline for the study described in Examples 13 and 14.

[0183] Figures 14A-14F show the effect of prophylactic CD20xCD3-mediated B cell depletion on serum anti-AAV8 IgM (Figure 14A and Figure 14D), IgG (Figure 14B and Figure 14E), and neutralizing antibody (nAb) (Figure 14C and Figure 14F) titers in cynomolgus macaques.

[0184] Figures 15A-15C show the effect of prophylactic CD20xCD3-mediated B cell depletion on AAV transduction (Figure 15A) and transgene expression (Figures 15B-15C) following AAV vector re-administration in cynomolgus macaques. DETAILED DESCRIPTION

[0185] The present disclosure provides, among other things, a distinct B cell immunosuppression approach that enables AAV vector re-transduction at levels equal to seronegative animals by depleting pre-existing nAbs (e.g., via combined plasma cell and immunoglobulin depletion). Long-lived plasma cells (LLPC) mediate constitutive antibody production to most antigens and are a likely reservoir of persistent anti-AAV antibody immunity. The present disclosure was made in part based on the discovery that pre-existing anti-AAV nAbs could be directly eliminated in vivo by LLPC depletion with linvoseltamab, a fully-human T cell-bridging bispecific antibody targeting B cell maturation antigen (BCMA) and CD3 (anti-BCMAxCD3 bispecific antibody), either alone or in combination with B cell depletion (to eliminate non-LLPC sources of anti-AAV nAbs) and / or FcRn blockade (to accelerate serum IgG clearance). Further, in AAV-naTve patients, prophylactic transient B cell depletion before and during AAV treatment with a bispecific antibody targeting CD20 and CD3 (anti-CD20xCD3 bispecific antibody) alone could be used to prevent an antibody response (e.g., the generation of anti-AAV nAbs), thereby allowing for effective AAV re-dosing. Definitions

[0186] Before the present invention is described, it is to be understood that the invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0187] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0188] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0189] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including multispecific antibodies, e.g., bispecific antibodies.

[0190] The term "antibody," as used herein, refers to an antigen-binding molecule or molecular complex comprising a set of complementarity determining regions (CDRs) that specifically bind to or interact with a particular antigen (e.g., BCMA, CD20, CD3). The term “antibody,” as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or Vh) and a heavy chain constant region. The heavy chain constant region comprises three domains, Ch1, Ch2 and Ch3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (Cl1). The Vh and Vl regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0191] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition (enhanced Chothia or Martin), the IMGT definition, and the Honneger definition (AHo). In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Chothia et al., J Mol Biol (1987), 4:901-17; Al-Lazikani et al., J. Mol. Biol. 273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989); see also, Dondelinger et al., Front. Immunol. (2018), 9:2278, doi:10.3389 / fimmu.2018.02278. Public databases are also available for identifying CDR sequences within an antibody.

[0192] The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, "antigen-binding domain," and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0193] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc ), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0194] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL orVL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric Vh or Vl domain.

[0195] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) Vl-Ch3; (xi) VL-CH1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) Vl-Ch2-Ch3; and (xiv) Vl-Cl. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigenbinding fragment of an antibody may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0196] The term "antibody," as used herein, also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen

[0197] Any multispecific antibody format may be adapted for use in the context of an antibody or antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art. For example, the present disclosure includes bispecific antibodies wherein one arm of an immunoglobulin is specific for an epitope of BCMA or CD20 and the other arm of the immunoglobulin is specific for an epitope of CD3. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see, e.g., Klein etal. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0198] The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0199] The term “recombinant antibody,” as used herein, is intended to include all antibodies that are prepared, expressed, created, or isolated by recombinant means. The term includes, but is not limited to, antibodies expressed using a recombinant expression vector transfected into a host cell (e.g., Chinese hamster ovary (CHO) cell) or cellular expression system, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies isolated from a non-human animal (e.g., a mouse, such as a mouse that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the Vh and Vl regions of the recombinant antibodies are sequences that, while derived from and related to human germline Vh and Vl sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0200] An "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody." An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0201] The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10'6 M or less, e.g., 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, or 10-12 M (a smaller Kd denotes a tighter binding). Methods for determining whether an antibody specifically binds to an antigen are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., BIACORE™), bio-layer interferometry assay (e.g., Octet® HTX biosensor), solution-affinity ELISA, and the like. In some embodiments, specific binding is measured in a surface plasmon resonance assay, e.g., at 25°C or 37°C. An antibody or antigen-binding fragment that specifically binds an antigen from one species may or may not have crossreactivity to other antigens, such as an orthologous antigen from another species.

[0202] The term "KD," as used herein, refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0203] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Cytiva, Marlborough, MA).

[0204] The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be either linear or discontinuous (e.g., conformational). A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups and, in some embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. An epitope typically includes at least 3, and more usually, e.g., at least 5 or at least 8-10 amino acids, in a unique spatial conformation.

[0205] Methods for determining the epitope of an antigen-binding protein, e.g., an antibody or antigen-binding fragment, include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mol Biol 2004, 248:443-463), peptide cleavage analysis, crystallographic studies, and nuclear magnetic resonance (NMR) analysis. In addition, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be employed (Tomer, Prot Sci 2000, 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or antigen-binding fragment) interacts is hydrogen / deuterium exchange detected by mass spectrometry (HDX). See, e.g., Ehring, Analytical Biochemistry 1999, 267:252-259; Engen and Smith, Anal Chem 2001, 73:256A-265A.

[0206] The term "competes," as used in reference to competing for binding, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment) that binds to an antigen and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigenbinding fragment) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in both orientations, i.e., a first antigen that binds an antigen and blocks binding of the antigen by a second antibody, and vice versa. Thus, in some embodiments, competition occurs in one such orientation. In some embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different epitopes, which may be overlapping or nonoverlapping, wherein binding of one antigen-binding protein inhibits or blocks the binding of the second antigen-binding protein, e.g., via steric hindrance. Competition between antigen-binding proteins may be measured by methods known in the art, e.g., by a real-time, label-free bio-layer interferometry assay.

[0207] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0208] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0209] In the context of the present disclosure, the term “neutralizing antibody” or “nAb” refers to an antibody that binds to a pathogen (e.g., a virus) and interferes with its ability to infect a cell. Non-limiting examples of neutralizing antibodies include antibodies that bind to a viral particle and inhibit successful transduction, e.g., one or more steps selected from binding, entry, trafficking to the nucleus, and transcription of the viral genome. Some neutralizing antibodies may block a virus at the post-entry step.

[0210] The term “immune response” refers to a response of a cell of the immune system (e.g., a B-cell, T-cell, macrophage or polymorphonucleocyte) to a stimulus such as an immunogen, e.g., antigen (e.g., a viral antigen). Active immune responses can involve differentiation and proliferation of immunocompetent cells, which leads to synthesis of antibodies or the development of cell-mediated reactivity, or both. An active immune response can be mounted by the host after exposure to an antigen (e.g., by infection or by vaccination). Active immune response can be contrasted with passive immunity, which can be acquired through the transfer of substances such as, e.g., an antibody, transfer factor, thymic graft, and / or cytokines, from an actively immunized host to a non-immune host.

[0211] The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted and other elements added without sacrificing the necessary expression.

[0212] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.

[0213] The terms “viral element” and “viral component” are used herein to refer to viral genes (e.g., genes encoding polymerase or structural proteins) or other elements of the viral genome (e.g., packaging signals, regulatory elements, LTRs, ITRs, etc.).

[0214] The term "capsid protein,” “Cap protein,” and the like, includes a protein that is part of the capsid of the virus.

[0215] The term “isolated” with respect to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or organism components that may normally be present in situ, up to and including a substantially pure preparation of the protein, nucleic acid, or cell. The term “isolated” may include proteins and nucleic acids that have no naturally occurring counterpart or proteins or nucleic acids that have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids. The term “isolated” may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular components or organism components with which they are naturally accompanied (e.g., but not limited to, other cellular proteins, nucleic acids, or cellular or extracellular components).

[0216] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by a heterologous promoter not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0217] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0218] A constitutive promoter is one that is active in all tissues or particular tissues at all developing stages. Examples of constitutive promoters include the human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEF1a), mouse elongation factor 1 alpha (mEF1a), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin promoters.

[0219] Examples of inducible promoters include, for example, chemically regulated promoters and physically-regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline (tet)-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid-regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically-regulated promoters include, for example, temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter ora light-repressible promoter).

[0220] Tissue-specific promoters can be, for example, neuron-specific promoters or glial-specific promoters or muscle-specific promoters.

[0221] Developmentally-regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.

[0222] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0223] The term "in vitro” includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term "in vivo” includes natural environments (e.g., a cell, organism, or body) and to processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells.

[0224] The term “fusogen” or "fusogenic molecule" is used herein to refer to any molecule that can trigger membrane fusion when present on the surface of a virus particle. A fusogen can be, for example, a protein (e.g., a viral glycoprotein) or a fragment, mutant or derivative thereof.

[0225] The term “oncolytic virus” is used herein to refer to a virus that is capable of infecting and replicating in a tumor cell such that the tumor cell may be killed. The oncolytic virus may be replication competent. As a non-limiting example, the oncolytic virus may comprise a rhabdovirus, i.e., any of a group of viruses comprising the family Rhabdoviridae, e.g., a vesicular stomatitis virus (VSV).

[0226] The term "T cell" is used herein in its broadest sense to refer to all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg), and natural killer (NK)-T cells.

[0227] “Retargeting” or “redirecting” may include a scenario in which a wildtype particle targets several cells within a tissue and / or several organs within an organism, and general targeting of the tissue or organs is reduced or abolished by insertion of the heterologous amino acid, and retargeting to more a specific cell in the tissue or a specific organ in the organism is achieved with the targeting ligand (e.g., via a targeting ligand) that binds a marker expressed by the specific cell. Such retargeting or redirecting may also include a scenario in which the wildtype particle targets a tissue, and targeting of the tissue is reduced to or abolished by insertion of the heterologous amino acid, and retargeting to a completely different tissue is achieved with the targeting ligand.

[0228] The term “wild type” or “wild-type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0229] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form or that are introduced into a cell from an outside source. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0230] “Specific binding pair,” “binding pair,” “protein:protein binding pair,” and the like, includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope; a covalent bond between e.g., proteins capable of forming isopeptide bonds; split inteins that recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal). In some embodiments, the term "cognate" refers to components that function together. Epitopes and cognate antibodies thereto, particularly epitopes that may also act as a detectable label (e.g., c-myc) are well-known in the art. Specific protein:protein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002; SpyTag:KTag; isopeptag:pilin C, SnoopTag:SnoopCatcher, etc., and variants thereof, e.g., SpyTag003:SpyCatcher003. Generally, a first member of a protein:protein binding pair refers to member of a protein:protein binding pair, which is generally less than 30 amino acids in length, and which forms a spontaneous covalent isopeptide bond with the second cognate protein, wherein the second cognate protein is generally larger, but may also be less than 30 amino acids in length such as in the SpyTag:KTag system.

[0231] The terms "substantial identity" and "substantially identical," as used with reference to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0232] As applied to polypeptides, the terms "substantial identity" and "substantially identical" mean that two peptide sequences, when optimally aligned, share at least about 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.

[0233] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 2000 supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. (See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402).

[0234] A "variant" of a polypeptide, such an immunoglobulin, VH, VL, heavy chain, light chain, or CDR comprising an amino acid sequence specifically set forth herein, refers to a polypeptide comprising an amino acid sequence that is at least about 70%-99.9% (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to the reference polypeptide sequence (e.g., as set forth in the sequence listing below), when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, a variant of a polypeptide includes a polypeptide having the amino acid sequence of a reference polypeptide sequence (e.g., as set forth in the sequence listing below) but for one or more (e.g., 1 to 10, or less than 20, or less than 10) missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions.

[0235] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0236] The phrase “pharmaceutically acceptable” as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0237] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0238] An “individual” or “subject” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human. Plasma Cell Depleting Agents

[0239] In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of a plasma cell depleting agent to a subject in need thereof. As used herein, a “plasma cell depleting agent” refers to any molecule capable of specifically binding to a surface antigen on plasma cells and killing or depleting said plasma cell.

[0240] The plasma cell depleting agents can be administered to a subject in need thereof either alone or in combination with a B cell depleting agent, and / or an immunoglobulin depleting agent. In various aspects, a plasma cell depleting agent may be combined or administered in combination with a B cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, immunoadsorption, and / or an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein. In some embodiments, the plasma cell depleting agent of the present disclosure is capable of depleting plasma cells including, without limitation, long-lived plasma cells (LLPCs). In some embodiments, a plasma cell depleting agent is administered to a subject having a pre-existing immunity against an immunogen (e.g. an immunogenic delivery vehicle such as, e.g., AAV)

[0241] In some embodiments, the plasma cell depleting agent can be an antibody, a small molecule compound, a nucleic acid, a polypeptide, or a functional fragment or variant thereof. Non-limiting examples of suitable plasma cell depleting agents include B cell maturation antigen (BCMA) targeting agents (described elsewhere herein), proteasome inhibitors [e.g., bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Niniaro)], histone deacetylase inhibitors [e.g., panobinostat (Farydak)], B-cell activating factor (BAFF; also referred to as BLyS, TALL-1, or CD257) inhibitors (e.g., anti-BAFF antibodies such as belimumab, tabalumab, AMG570; or anti-BAFF receptor antibodies such as ianalumab), proliferation-inducing ligand (APRIL; also referred to as TNFSF13 or CD256) inhibitors (e.g., anti-APRIL antibodies such as BION-1301 or VIS624), G protein-coupled receptor, class C, group 5, member D (GPRC5D) inhibitors (e.g., anti-GPRC5D antibodies, anti-GPRC5DxCD3 bispecific antibodies such as talquetamab), Fc receptor homolog 5 (FcRH5; also referred to as FcRL5, IRTA2, or CD307) inhibitors (e.g., anti-FcRH5 antibodies, anti-FcRH5 xCD3 bispecific antibodies such as Cevostamab), and cluster of differentiation 38 (CD38; also referred to as CADPR1 or ADPRC1) inhibitors (e.g., anti-CD38 antibodies).

[0242] In some embodiments, the plasma cell depleting agents used in the methods disclosed herein are BCMA targeting agents. As used herein, the term “BCMA targeting agent” refers to any molecule capable of binding specifically to BCMA that is expressed on the surface of a cell, e.g, a cell in a subject, thus targeting said cell for destruction. BCMA is expressed exclusively in B-cell lineage cells, particularly in the interfollicular region of the germinal center, as well as on plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is required for optimal survival of long-lived plasma cells (LLPCs) in the bone marrow. Thus, a BCMA targeting agent binds to BCMA expressed on a plasma cell surface and mediates killing or depletion of cells that express BCMA (plasma cell depletion). In some embodiments, a BCMA targeting agent comprises a binding moiety that binds to plasma cellsurface-expressed BCMA (an antigen-binding moiety or antigen-binding fragment thereof) and a moiety that facilitates killing of said plasma cell. In some embodiments, the plasma cell-surface-expressed BCMA-binding moiety is an antibody or antigen-binding fragment thereof that binds specifically to BCMA. Such a BCMA-binding moiety can be linked (e.g., covalently bound) to a moiety that facilitates killing or destruction of the targeted plasma cell. The moiety that facilitates targeted killing of the bound plasma cell may be a molecule that directly kills the targeted cell (e.g., a cytotoxic agent) or may be a protein or fragment thereof that mediates killing of the targeted cell, e.g., by an immune cell, e.g., a T-cell. In the context of the present disclosure, the term “BCMA targeting agent” includes, but is not limited to, anti-BCMA antibodies that are conjugated to a therapeutic agent such as a cytotoxic drug (“BCMA ADC” or “anti-BCMA ADC”, e.g., Belantamab Mafodotin (GSK2857916), MEDI2228, HDP-101), chimeric antigenic receptors (CARs) that bind specifically to BCMA, (“BCMA CAR” or “anti-BCMA CAR”) and anti-BCMAxCD3 bispecific antibodies (e.g., linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), andTNB384B).

[0243] In some embodiments, the BCMA targeting agent used in the context of the disclosed methods is an antibody-drug conjugate (ADC) comprising an anti-BCMA antibody and a cytotoxic drug. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof and the cytotoxic agent are covalently attached via a linker. In general terms, the ADCs comprise: A-[L-P]y, in which A is an antigen-binding molecule, e.g., an anti-BCMA antibody, ora fragment thereof, L is a linker, P is the payload or therapeutic moiety (e.g., cytotoxic agent), and y is an integer from 1 to 30. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming ADCs are known in the art. Non-limiting examples of suitable cytotoxic agents that can be conjugated to anti-BCMA antibodies for use in the disclosed methods are auristatin such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), a tubulysin such as TUB-OH orTUB-OMOM, a tomaymycin derivative, a dolastatin derivative, or a maytansinoid such as DM1 or DM4. In some exemplary embodiments, an anti-BCMA ADC used in the present methods comprises the HCVR, LCVR and / or CDR amino acid sequences of any of the anti-BCMA antigen-binding molecules disclosed herein.

[0244] Other anti-BCMA ADCs that can be used in the context of the methods of the present disclosure include, e.g., the ADCs referred to and known in the art as Belantamab Mafodotin (GSK2857916), AMG224, HDP-101, MEDI2228, and TBL-CLN1, or any of the anti-BCMA ADCs set forth, e.g., in International Patent Publications WO2011 / 108008, WO2014 / 089335, WO2017 / 093942, WO2017 / 143069, or WO2019 / 025983. The portions of the publications cited herein that identify anti-BCMA ADCs are hereby incorporated by reference.

[0245] In some embodiments, the BCMA targeting agent used in the context of the disclosed methods is a chimeric antigen receptor (CAR) that binds specifically to BCMA (“BCMA CAR”). Generally, a “chimeric antigen receptor” (CAR) exhibits a specific anti-target cellular immune activity and comprises a binding domain against a component present on the target cell, for example an antibody-based specificity for a desired antigen (e.g., BCMA on plasma cell), and a T cell receptor-activating intracellular domain. CARs typically comprise an extracellular single chain antibody-binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity. In certain embodiments, the BCMA CAR or antigen-binding fragment thereof comprises a HCVR, LCVR, and / or CDRs comprising the amino acid sequences of any of the antibodies set forth in US Patent Publication No. US 2020 / 0023010, which is hereby incorporated by reference in its entirety. In some exemplary embodiments, an anti-BCMA CAR used in the present methods comprises the HCVR, LCVR and / or CDR amino acid sequences of any of the anti-BCMA antigen-binding molecules disclosed herein.

[0246] Other anti-BCMA CARs that can be used in the context of the methods of the present disclosure include, e.g., the CARs referred to and known in the art as bb2121, LCAR-B38M, and 4C8A, or any of the anti-BCMA CARs set forth, e.g., in patent publications WO2015 / 052538, WO2015 / 052536, WO2016 / 094304, WO2016 / 166630, WO2016 / 151315, WO2016 / 130598, WO2017 / 183418, WO2017 / 173256, WO2017211900, WO2017 / 130223, WO2018 / 229492, WO2018 / 085690, WO2018 / 151836, WO2018 / 028647, WO2019 / 006072. The portions of the publications cited herein that identify anti-BCMA CARs are hereby incorporated by reference.

[0247] In some exemplary embodiments, the BCMA targeting agent used in the disclosed methods is a multispecific (e.g., bispecific) antibody, or a functional fragment thereof, that specifically binds B cell maturation antigen (BCMA) and CD3 (e.g., an anti-BCMAxCD3 bispecific antibody). The anti-BCMAxCD3 multispecific (e.g., bispecific) antibodies are useful for specific targeting and T-cell-mediated killing of cells that express BCMA. The terms “antibody,” “antigen-binding fragment,” “human antibody,” “recombinant antibody,” and other related terminology are defined above. In the context of anti-BCMAxCD3 antibodies and antigenbinding fragments thereof, the present disclosure includes the use of bispecific antibodies wherein one arm of an immunoglobulin is specific for BCMA or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target (e.g., CD3 on T-cells). Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, lgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mabe bispecific formats (see, e.g., Klein etal. 2012, mAbs 4(6):653-663, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate sitespecific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chern. Soc., 2013, 135(1):340-46).

[0248] An anti-BCMAxCD3 bispecific antibody, or functional fragment thereof, may comprise any of various anti-BCMAxCD3 bispecific antibodies, or functional fragments thereof, disclosed herein, or any other such anti-BCMAxCD3 bispecific antibodies, or functional fragments thereof, known to persons of ordinary skill in the art (e.g., linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), andTNB384B). CD3 Antigen-Binding Molecules

[0249] The term “CD3,” as used herein, refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the dimeric association of two of four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta). CD3 is required for T cell activation.

[0250] As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded major histocompatibility complex (MHC) molecules. Thus, bispecific antigen-binding molecules that are capable of binding both CD3 and another antigen (e.g., CD20 or BCMA) would be useful in settings in which specific targeting and T cell-mediated killing of cells that express the non-CD3 antigen (e.g., CD20 or BCMA) is desired.

[0251] The antibodies and antigen-binding fragments of the present invention may bind soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.

[0252] As used herein, the expression “cell surface-expressed CD3” means one or more CD3 protein(s) that is / are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. “Cell surface-expressed CD3” includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell. The expression “cell surface-expressed CD3” includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The expression “cell surface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma) that is expressed by itself, without other CD3 chain types, on the surface of a cell. A “cell surface-expressed CD3” can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein. Alternatively, “cell surface-expressed CD3” can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0253] As used herein, the expression “anti-CD3 antibody” includes both monovalent antibodies with a single specificity, as well as bispecific antibodies comprising one arm that binds CD3 and another arm that binds a different antigen, wherein the anti-CD3 arm comprises any of the HCVR / LCVR or CDR sequences, or functional fragments thereof, as set forth in Table 1 or Table 2 herein. Examples of anti-CD3 bispecific antibodies are described elsewhere herein. Exemplary anti-CD3 antibodies are also described in PCT International Application No. PCT / US2013 / 060511, which is herein incorporated by reference in its entirety.

[0254] The present disclosure includes bispecific antibodies and functional fragments thereof that bind human CD3 with high affinity. The present disclosure also includes bispecific antibodies and functional fragments thereof that bind human CD3 with medium or low affinity, depending on the therapeutic context and particular targeting properties that are desired. For example, in the context of a bispecific antigen-binding molecule, wherein one arm binds CD3 and a second arm binds another antigen (e.g., CD20 or BCMA), it may be desirable for the second arm to bind the non-CD3 (e.g., CD20 or BCMA) antigen with high affinity while the anti-CD3 arm binds CD3 with only moderate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the non-CD3 (e.g., CD20 or BCMA) antigen may be achieved while avoiding general / untargeted CD3 binding and the consequent adverse side effects associated therewith.

[0255] In certain embodiments, the anti-CD3 antibodies induce T cell proliferation with an ECso value of less than about 0.33 pM, as measured by an in vitro T cell proliferation assay (e.g., assessing the proliferation of Jurkat cells or PBMCs in the presence of anti-CD3 antibodies). In certain embodiments, the anti-CD3 antibodies induce T cell proliferation (eg., Jurkat cell proliferation and / or PBMC proliferation) with an EC50 value of less than about 0.32 pM, less than about 0.31 pM, less than about 0.30 pM, less than about 0.28 pM, less than about 0.26 pM, less than about 0.24 pM, less than about 0.22 pM, or less than about 0.20 pM, as measured by an in vitro T cell proliferation assay. BCMAxCD3 Antigen-Binding Molecules

[0256] The present disclosure provides antigen-binding molecules including multispecific (e.g., bispecific) antibodies that specifically bind B cell maturation antigen (BCMA) and CD3 (e.g., an anti-BCMAxCD3 bispecific antibody). In some embodiments, the antigen-binding molecule is a multispecific (e.g., bispecific) antibody. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt etal., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. In some embodiments, the multispecific antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity. In some embodiments, the multispecific antibody contains an antigen-binding domain that is specific for BCMA and an antigen-binding domain that is specific for CD3.

[0257] In some embodiments, the anti-BCMAxCD3 bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that binds an epitope of BCMA (e.g., human BCMA), and a second antigen-binding domain (D2) that binds an epitope of CD3 (e.g., human CD3).

[0258] In some exemplary embodiments, the anti-BCMAxCD3 bispecific antibody, or antigenbinding fragment thereof, comprises a heavy chain variable region (HCVR), light chain variable region (LCVR), and / or complementarity determining regions (CDRs) comprising the amino acid sequences of any of the anti-BCMAxCD3 antibodies set forth in US Patent No. 11,384,153 and US Patent Publication No. 2020 / 0345843, which are hereby incorporated by reference in their entireties.

[0259] In some exemplary embodiments, an anti-BCMAxCD3 bispecific antibody or antigenbinding fragment thereof that can be used in the context of the present disclosure comprising a HCVR, a LCVR, and / or CDRs comprising the amino acid sequences of REGN5458 or REGN5459 as set forth in Table 1 below. Table 1. Amino Acid Sequences of Exemplary Anti-BCMA* CD3 Bispecific Antibodies Anti-BCMA First Antigen-Binding Domain Anti-CD3 Second Antigen-Binding Domain Common Light Chain Variable Region Bispecific antibody identifier HCV R HCDR 1 HCDR 2 HCDR 3 HCV R HCDR 1 HCDR 2 HCDR 3 LCV R LCDR 1 LCDR 2 LCDR 3 REGN545 8 2 4 6 8 26 28 30 32 18 20 22 24 REGN545 9 2 4 6 8 34 36 38 40 18 20 22 24

[0260] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof that can be used the present disclosure comprises: (a) a first antigen binding domain that binds specifically to BCMA; and (b) a second antigen-binding domain that binds specifically to CD3. In one embodiment, the anti-BCMA antigen-binding domain comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the first antigenbinding domain comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 22; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0261] In one embodiment, the second antigen-binding domain comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 34 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second antigen-binding domain comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 28 or 36; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 30 or 38; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 32 or 40; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 22; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0262] In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24; and (b) a second antigen binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 28, 30, and 32, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24. In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 2 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 26 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18.

[0263] In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24; and (b) a second antigen binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 40, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24. In one embodiment, the anti-BCMA / anti-CD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 2 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 34 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18.

[0264] Exemplary anti-BCMAxCD3 bispecific antibodies include the fully human bispecific antibodies known as REGN5458 and REGN5459. See, e.g., WO 2020 / 018820, US 2020 / 0024356, US 2022 / 0306758, and US 11,384,153, each of which is herein incorporated by reference. According to certain exemplary embodiments, the methods of the present disclosure comprise the use of REGN5458 or REGN5459, or a bioequivalent thereof. As used herein, the term “bioequivalent” with respect to anti-BCMAxCD3 antibodies refers to antibodies or BCMAxCD3 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives having a rate and / or extent of absorption that does not show a significant difference with that of a reference antibody (e.g., REGN5458 or REGN5459) when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose; the term “bioequivalent” also includes antigen-binding proteins that bind to BCMA / CD3 and do not have clinically meaningful differences with the reference antibody (e.g., REGN5458 or REGN5459) with respect to safety, purity, and / or potency.

[0265] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 28, 30, and 32, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0266] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 40, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0267] The present disclosure also includes variants of the anti-BCMAxCD3 antibodies described herein comprising any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative amino acid substitutions. For example, the present disclosure includes use of anti-BCMAxCD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an anti-BCMAxCD3 antibody having HCVR, LCVR, and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0268] Other anti-BCMAxCD3 antibodies that can be used in the methods of the present disclosure include, e.g., the antibodies referred to and known in the art as pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B, or any of the anti-BCMAxCD3 antibodies set forth, e.g., in International Patent Publications WO2013 / 072415, WO2014 / 140248, WO2014 / 122144, WO2016 / 166629, WO2016 / 079177, WO2016 / 020332, WO2017031104, WO2017 / 223111, WO2017 / 134134, WO2018 / 083204, or WO2018 / 201051. The portions of the publications cited herein that identify anti-BCMAxCD3 antibodies are hereby incorporated by reference.

[0269] In some embodiments, the CDRs disclosed herein are identified according to the Kabat definition. In some embodiments, the CDRs are identified according to the Chothia definition. In some embodiments, the CDRs are identified according to the AbM definition. In some embodiments, the CDRs are identified according to the IMGT definition.

[0270] The bispecific antigen-binding molecules disclosed herein may be bispecific antibodies. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype lgG1. In some cases, the human IgG heavy chain constant region is isotype lgG4. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that reduces binding to an Fc receptor. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn). In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

[0271] In some embodiments, the heavy chain constant region attached to the HCVR of the first antigen-binding domain or the heavy chain constant region attached to the HCVR of the second antigen-binding domain, but not both, contains an amino acid modification that reduces Protein A binding relative to a heavy chain of the same isotype without the modification. In some cases, the modification comprises a H435R substitution (EU numbering) in a heavy chain of isotype lgG1 or lgG4. In some cases, the modification comprises a H435R substitution and a Y436F substitution (EU numbering) in a heavy chain of isotype IgG 1 or lgG4.

[0272] In some embodiments, the antibody comprises a first heavy chain containing the HCVR of the first antigen-binding domain and a second heavy chain containing the HCVR of the second antigen-binding domain, wherein the first heavy chain comprises residues 1-450 of the amino acid sequence of SEQ ID NO: 41 and the second heavy chain comprises residues 1-449 of the amino acid sequence of SEQ ID NO: 42.

[0273] In some embodiments, the antibody comprises a common light chain containing the LCVR of the first and second antigen-binding domains, wherein the common light chain comprises the amino acid sequence of SEQ ID NO: 43.

[0274] In some embodiments, the anti-BCMAxCD3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 42, and a common light chain comprising the amino acid sequence of SEQ ID NO: 43. In some cases, the mature form of the antibody may not include the C-terminal lysine residues of SEQ ID NOs: 41 and 42. Thus, in some cases the anti-BCMA binding arm comprises a heavy chain comprising residues 1-450 of SEQ ID NO: 41, and the anti-CD3 binding arm comprises a heavy chain comprising residues 1-449 of SEQ ID NO: 42.

[0275] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigenbinding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes lgG1, lgG2, lgG3, and lgG4, as well as any allotype within each isotype group.

[0276] In some embodiments, a bispecific antigen-binding molecule of the present disclosure comprises two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1 / lgG1, lgG2 / lgG2, lgG4 / lgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1 / lgG2, lgG1 / lgG4, lgG2 / lgG4, etc.

[0277] In some embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length, containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. B Cell Depleting Agents

[0278] In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of a B cell depleting agent to a subject in need thereof. As used herein, a “B cell depleting agent” refers to any molecule capable of specifically binding to a surface antigen on B cells and killing or depleting said B cell. Thus, in general, a B cell depleting agent can be any agent that binds to a B cell surface molecule. In some embodiments, the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

[0279] In various aspects, the present disclosure provides B cell depleting agents, which may be administered to a subject in need thereof, e.g., either alone or combined with, or administered in combination with, a plasma cell depleting agent (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof), an immunoglobulin depleting agent (e.g., an FcFn blocker such as, e.g., efgartigimod), and / or an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV). In some embodiments plasmapheresis, therapeutic plasma exchange, and / or immunoadsorption may be further combined with the administering of the B cell depleting agent, the plasma cell depleting agent, the immunoglobulin depleting agent, and / or the immunogen. In some embodiments, the subject does not have a pre-existing immunity against the immunogen.

[0280] In some embodiments, a B cell depleting agent may be administered alone (e.g., as a monotherapy, in the absence of the administration of any other additional immunomodulators [e.g., plasma cell depleting agents, immunoglobulin depleting agents], and optionally, combined with, or administered in combination with, an immunogen) to a subject in need thereof, e.g., a subject without a pre-existing immunity against an immunogen (e.g., an immunogen to be administered to the subject e.g., an immunogenic delivery vehicle such as, e.g., AAV). In some embodiments, the B cell depleting agent may be administered alone to a subject who is immunologically naive to an immunogen to be administered to the subject (e.g., AAV). In some embodiments, the B cell depleting agent may be administered alone to an AAV seronegative subject, and the subject is further administered an immunogen (e.g., AAV). In some embodiments, a B cell depleting agent may be useful as a prophylactic treatment to prevent or suppress an immune response (e.g., an anti-AAV IgG, IgM, and / or nAb response) to an immunogen (e.g., AAV) in a subject in need thereof (e.g., a subject without a pre-existing immunity to the immunogen).

[0281] In some embodiments, the suppression or prevention of an immune response (e.g., an anti-AAV IgG, IgM, and / or nAb response) to an immunogen in a subject (e.g., a subject without a pre-existing immunity to the immunogen) can be achieved by administering a B cell depleting agent described herein (e.g., an anti-CD20xCD3 bispecific antibody or a functional fragment thereof). Administration of the B cell depleting agent to the subject can suppress or prevent the immune response in the subject following the initial dosing and / or re-dosing of an immunogen (e.g., post-AAV dosing and / or re-dosing). In some embodiments, an immune response may be suppressed in a subject following AAV dosing and / or re-dosing. The immune response may be suppressed by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more, e.g., relative to an immune response in a subject receiving no immunomodulation treatment or treatment with a conventional anti-CD20 therapeutic alone (e.g., rituximab, or derivatives or equivalents thereof). The immune response may be suppressed by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The immune response may be suppressed by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100%. In some embodiments, the immune response is prevented. In some embodiments, an immune response may be suppressed or prevented in a subject following AAV dosing and / or re-dosing in the subject to achieve levels equivalent to, or even below, those of an AAV-naTve subject. In some embodiments, a B cell depleting agent is sufficient to enable effective re-dosing of an immunogen to a subject. The B cell depleting agent can be administered to the subject prior to the re-dosing of the immunogen any number of times and can be used to maintain a suppressed immune response to the immunogen in the subject for any period of time thereafter.

[0282] In some embodiments, a B cell depleting agent is capable of suppressing an antiimmunogen response (e.g., an anti-AAV response) in a subject, and the anti-immunogen response is mounted by the subject in response to repeated doses of the immunogen (e.g. AAV).

[0283] It is contemplated that a B cell depleting agent may be used in the suppression or prevention of an anti-immunogen antibody response (e.g., an anti-AAV antibody response) in a subject, and the suppression or prevention of the anti-immunogen antibody response involves B cell depletion in primary and / or secondary lymphoid tissues. Non-limiting examples of primary lymphoid tissues include bone marrow and thymus. In some embodiments, the compositions and methods of the disclosure encompass B cell depletion in secondary lymphoid tissues, for example, and without limitation, spleen and / or lymph nodes. In some embodiments, the compositions and methods of the disclosure relate to B cell depletion in lymph nodes, which is achieved by a B cell depleting agent described herein.

[0284] In some embodiments, the present disclosure provides B cell depleting agents combined with, or administered in combination with, plasma cell depleting agents (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof) described herein to subjects, e.g., subjects with or without a pre-existing immunity against an immunogen (i.e., an immunogen administered to the subject, e.g., an immunogenic delivery vehicle such as, e.g., AAV). In some embodiments, the B cell depleting agent may be administered in combination with a plasma cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, immunoadsorption, and / or an immunogen (e.g., an immunogenic delivery vehicle) disclosed herein. In some embodiments, the B cell depleting agent may be administered to subjects without a pre-existing immunity against an immunogen (i.e., an immunogen to be administered to the subject, e.g., an immunogenic delivery vehicle such as, e.g., AAV) not only alone, but also in combination with a plasma cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, or immunoadsorption, and / or an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein.

[0285] In some embodiments, the B cell depleting agent is an agent that directly targets a B cell, e.g., an agent that binds to a B cell surface molecule. In some embodiments, the B cell depleting agent causes a reduction in the number of B cells in a subject (e.g., in a blood sample taken from the subject). In some embodiments, a B cell depleting agent may be useful for, e.g., eliminating non-plasma cell (e.g. non-long-lived plasma cell [LLPC] sources of immunogen (e.g., anti-AAV) nAbs. In some embodiments, a B cell depleting agent may be useful for, e.g., preventing formation of non-plasma cell (e.g. non-long-lived plasma cell [LLPC] sources of immunogen (e.g., anti-AAV) nAbs (e.g., in AAV-naTve patients). In some embodiments, the B cell depleting agent may capture a wider range of AAV-specific B cells and plasma cells that may not express high levels of BCMA (e.g., committed memory B cells and early plasmablasts).

[0286] In some embodiments, the B cell depleting agent comprises an anti-CD19 antibody (e.g., MEDI-551, tefasitamab, Inebilizumab, loncastuximab), an anti-CD20 antibody (e.g., rituximab, ocrelizumab, obinutuzumab, ublituximab, or ofatumumab), an anti-CD22 antibody (e.g., epratuzumab), an anti-CD79 antibody (e.g., polatuzumab), a bispecific CD20xCD3 B cell depleting antibody (e.g. odronextamab, glofitamab, mosunetuzumab, epcoritamab), a bispecific CD19xCD3 antibody (e.g., blinatumomab), a bispecific CD22xCD3 antibody (e.g., inotuzumab), or functional fragments thereof, or any combination thereof.

[0287] In some embodiments, the B cell depleting agent is an agent that indirectly targets a B cell, e.g., by targeting a B cell survival factor. In some embodiments, the B cell depleting agent is a BLyS / BAFF inhibitor (e.g., belimumab, lanalumab, BR3-Fc, AMG-570, or AMG-623), an APRIL inhibitor (e.g., telitacicept, atacicept), or a BLyS receptor 3 / BAFF receptor inhibitor (e.g., anti-BR3), or any combination thereof.

[0288] In some embodiments, the B cell depleting agent is selected from anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD79 antibodies, multispecific antibodies combining two or more of any of said antibody specificities, multispecific antibodies combining any of said antibody specificities with anti-CD3 antibodies, functional fragments of any of said antibodies, and any combinations thereof. In some embodiments, the B cell depleting agent comprises an anti-CD20 antibody or a functional fragment thereof and an anti-CD19 antibody or a functional fragment thereof. In certain embodiments, the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof. In some embodiments, a multispecific anti-CD20 antibody or functional fragment thereof of the present disclosure targets CD20 and CD19. In some embodiments the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD19xCD20 bispecific antibody, or functional fragment thereof.

[0289] In some embodiments, the B cell depleting agent comprises anti-CD19 and anti-CD20 antibodies (also referred to as “anti-CD19 / CD20 antibodies” herein), or functional fragments thereof, disclosed herein.

[0290] In a specific embodiment, the B cell depleting agent comprises a bispecific antibody that specifically binds CD3 and CD19. Such antibodies may be referred to herein as, e.g., “anti-CD19 / anti-CD3,” or “anti-CD19xCD3” or “CD19xCD3” bispecific antibodies, or other similar terminology.

[0291] In a specific embodiment, the B cell depleting agent comprises a bispecific antibody that specifically binds CD3 and CD20. Such antibodies may be referred to herein as, e.g., “anti CD20 / anti-CD3,” or “anti-CD20xCD3” or “CD20xCD3” bispecific antibodies, or other similar terminology.

[0292] As used herein, the expression “bispecific antibody” refers to an immunoglobulin protein comprising at least a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds a first antigen (e.g., CD20), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., CD3). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each comprising three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix “A” and the CDRs of the second antigen-binding domain may be designated with the prefix “B”. Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1, B-HCDR2, and B-HCDR3.

[0293] The first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerizing domain. As used herein, a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. In the context of the present invention, the multimerizing component is an Fc portion of an immunoglobulin (comprising a Ch2-Ch3 domain), e.g., an Fc domain of an IgG selected from the isotypes lgG1, IgG2, lgG3, and lgG4, as well as any allotype within each isotype group.

[0294] Bispecific antibodies of the present invention typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1 / lgG1, lgG2 / lgG2, lgG4 / lgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1 / lgG2, lgG1 / lgG4, lgG2 / lgG4, etc.

[0295] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).

[0296] In the context of bispecific antibodies of the present invention, Fc domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigenbinding molecule comprises a modification in a Ch2 or a Ch3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in US 2015 / 0266966, incorporated herein in its entirety.

[0297] The present invention also includes bispecific antibodies comprising a first Ch3 domain and a second Ig Ch3 domain, wherein the first and second Ig Ch3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig Ch3 domain binds Protein A and the second Ig Ch3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second Ch3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second Ch3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 by EU) in the case of lgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 by EU) in the case of lgG4 antibodies.

[0298] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a Ch2 sequence derived from a human IgG 1, human lgG2 or human lgG4 Ch2 region, and part or all of a Ch3 sequence derived from a human lgG1, human lgG2 or human lgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human lgG1, a human lgG2 or a human lgG4 hinge region, combined with a “lower hinge” sequence, derived from a human IgG 1, a human lgG2 or a human lgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG4 Cn1]-[lgG4 upper hinge]-[lgG2 lower hinge]-[lgG4 Ch2]-[lgG4 Ch3], Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG1 CHl]-[lgG1 upper hinge]-[lgG2 lower hinge]-[lgG4 Ch2] [IgG 1 Ch3], These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in US Patent Publication No. 2014 / 0243504, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function. CD20xCD3 Antigen-Binding Molecules

[0299] The term “CD20,” as used herein, refers to an antigen which is expressed on B cells and which consists of a non-glycosylated phosphoprotein expressed on the cell membranes of mature B cells. The human CD20 protein can have the amino acid sequence as in NCBI Reference Sequence NP_690605.1. As used herein, the expression “anti-CD20 antibody” includes monovalent antibodies with a single specificity, such as RITUXAN® (rituximab), as described in U.S. Pat. No. 7,879,984. Exemplary anti-CD20 antibodies are also described in U.S. Pat. No. 7,879,984 and PCT International Application No. PCT / US2013 / 060511, filed on Sep. 19, 2013, each incorporated by reference herein.

[0300] In some exemplary embodiments, the CD20 targeting agent used in the disclosed methods is a multispecific (e.g., bispecific) antibody, or a functional fragment thereof, that specifically binds CD20 and CD3 (e.g., an anti-CD20xCD3 bispecific antibody). The anti-CD20xCD3 multispecific (e.g., bispecific) antibodies are useful for specific targeting and T-cell-mediated killing of cells that express CD20. The terms “antibody,” “antigen-binding fragment,” “human antibody,” “recombinant antibody,” and other related terminology are defined above. In the context of anti-CD20xCD3 antibodies and antigen-binding fragments thereof, the present disclosure includes the use of bispecific antibodies wherein one arm of an immunoglobulin is specific for CD20 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target (e.g., CD3 on T-cells). Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mabe bispecific formats (see, e.g., Klein et al. 2012, mAbs 4(6):653-663, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chern. Soc., 2013, 135(1):340-46).

[0301] The anti-CD20xCD3 bispecific antibodies are capable of simultaneously binding to human CD3 and human CD20. According to certain embodiments, the anti-CD20xCD3 bispecific antibodies specifically interact with cells that express CD3 and / or CD20. The extent to which the anti-CD20xCD3 bispecific antibodies binds cells that express CD3 and / or CD20 can be assessed by fluorescence activated cell sorting (FACS). In certain embodiments, the anti-CD20xCD3 bispecific antibodies specifically bind human T-cell lines which express CD3 (e.g., Jurkat), human B-cell lines which express CD20 (e.g., Raji), and primate T-cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]).

[0302] In some embodiments, the anti-CD20xCD3 bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that binds an epitope of CD20 (e.g., human CD20), and a second antigen-binding domain (D2) that binds an epitope of CD3 (e.g., human CD3).

[0303] According to certain exemplary embodiments of the present invention, the bispecific anti-CD20xCD3 antibody, or antigen-binding fragment thereof comprises heavy chain variable regions (A-HCVR and B-HCVR), light chain variable region (LCVR), and / or complementarity determining regions (CDRs) comprising any of the amino acid sequences of the bispecific anti-CD20xCD3 antibodies as set forth in US Patent Publication No. 20150266966, incorporated herein by reference in its entirety.

[0304] In certain exemplary embodiments, the bispecific anti-CD20xCD3 antibody or antigenbinding fragment thereof that can be used in the context of the methods of the present invention comprises: (a) a first antigen-binding arm comprising the heavy chain complementarity determining regions (A-HCDR1, A-HCDR2 and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 44 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding arm comprising the heavy chain CDRs (B-HCDR1, B-HCDR2 and B-HCDR3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 46 and the light chain CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO: 45. According to certain embodiments, the A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 47; the A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 48; the A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52; the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 53; the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 54; and the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 55. In yet other embodiments, the bispecific anti-CD20xCD3 antibody or antigen-binding fragment thereof comprises: (a) a first antigenbinding arm comprising a HCVR (A-HCVR) comprising SEQ ID NO: 44 and a LCVR comprising SEQ ID NO: 45; and (b) a second antigen-binding arm comprising a HCVR (B-HCVR) comprising SEQ ID NO: 46 and a LCVR comprising SEQ ID NO: 45.

[0305] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0306] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0307] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0308] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0309] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0310] Other bispecific anti-CD20xCD3 antibodies that can be used in the context of the methods of the present invention include, e.g., any of the antibodies as set forth in US 2014 / 0088295, US 2015 / 0166661, and US 2017 / 0174781, the disclosure of each of which is incorporated by reference in its entirely. An exemplary bispecific anti-CD20xCD3 antibody that can be used in the context of the methods of the present invention is the bispecific anti-CD20xCD3 antibody known as REGN1979 or bsAB1.

[0311] In some exemplary embodiments, an anti-CD20xCD3 bispecific antibody or antigenbinding fragment thereof that can be used in the context of the present disclosure comprising a HCVR, a LCVR, and / or CDRs comprising the amino acid sequences of REGN1979 as set forth in Table 2 below. Table 2. Amino Acid Sequences of Exemplary Anti-CD20*CD3 Bispecific Antibodies. Anti-CD20 First Antigen-Binding Domain Anti-CD3 Second Antigen-Binding Domain Common Light Chain Variable Region Bispecific antibody identifier HCV R HCDR 1 HCDR 2 HCDR 3 HCV R HCDR 1 HCDR 2 HCDR 3 LCV R LCDR 1 LCDR 2 LCDR 3 REGN19 79 44 47 48 49 46 53 54 55 45 50 51 52

[0312] In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof that can be used in the present disclosure comprises: (a) a first antigen binding domain that binds specifically to CD20; and (b) a second antigen-binding domain that binds specifically to CD3. In one embodiment, the anti-CD20 antigen-binding domain comprises the heavy chain complementarity determining regions (A-HCDRs) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 44 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the first antigenbinding domain comprises three HCDRs (A-HCDR1, A-HCDR2 and A-HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 47; the A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 48; the A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0313] In one embodiment, the second antigen-binding domain comprises the heavy chain complementarity determining regions (B-HCDRs) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 46 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the second antigen-binding domain comprises three HCDRs (B-HCDR1, B-HCDR2 and B-HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 53; the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 54; the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 55; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0314] In one embodiment, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises A-HCDR1, A-CDR2, and A-HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52; and (b) a second antigen binding domain that comprises B-HCDR1, B-HCDR2, and B-HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 53, 54, and 55, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52. In one embodiment, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a A-HCVR comprising the amino acid sequence of SEQ ID NO: 44 and a LCVR comprising the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises a B-HCVR comprising the amino acid sequence of SEQ ID NO: 46 and a LCVR comprising the amino acid sequence of SEQ ID NO: 45.

[0315] Exemplary anti-CD20xCD3 bispecific antibodies include the fully human bispecific antibody known as REGN1979. See, e.g., US 2014 / 0088295, US 2015 / 0166661, and US 2017 / 0174781, each of which is herein incorporated by reference. According to certain exemplary embodiments, the methods of the present disclosure comprise the use of REGN1979, or a bioequivalent thereof. As used herein, the term “bioequivalent” with respect to anti-CD20xCD3 antibodies refers to antibodies or CD20xCD3 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives having a rate and / or extent of absorption that does not show a significant difference with that of a reference antibody (e.g., REGN1979) when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose; the term “bioequivalent” also includes antigenbinding proteins that bind to CD20 / CD3 and do not have clinically meaningful differences with the reference antibody (e.g., REGN1979) with respect to safety, purity, and / or potency.

[0316] In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a A-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises a B-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 46 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (A-HCDR1, A-HCDR2 and A-HCDR3) comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively, and an A-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises three HCDRs (B-HCDR1, B-HCDR2 and B-HCDR3) comprising the amino acid sequences of SEQ ID NOs: 53, 54, and 55, respectively, and a B-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 46, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:45.

[0317] The present disclosure also includes variants of the anti-CD20xCD3 antibodies described herein comprising any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative amino acid substitutions. For example, the present disclosure includes use of anti-CD20xCD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an anti-CD20xCD3 antibody having HCVR, LCVR, and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0318] In some embodiments, the CDRs disclosed herein are identified according to the Kabat definition. In some embodiments, the CDRs are identified according to the Chothia definition. In some embodiments, the CDRs are identified according to the AbM definition. In some embodiments, the CDRs are identified according to the IMGT definition.

[0319] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region. In some embodiments, the human IgG heavy chain constant region is isotype lgG4 or lgG1. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that reduces binding to an Fc receptor. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn). In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR). Sequence Variants

[0320] The antigen-binding molecules of the present disclosure may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germ line sequences available from, for example, public antibody sequence databases. The antigen binding molecules of the present disclosure may comprise antigen binding fragments which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the Vh and / or Vl domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germ line sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germ line sequence while certain other residues that differ from the original germ line sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved, or enhanced antagonistic or agonistic biological properties, reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0321] The present disclosure also includes antigen-binding molecules wherein one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes antigen-binding molecules comprising an antigen-binding domain having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitution(s) relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an antibody having HCVR, LCVR and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet etal. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0322] The present disclosure also includes antigen-binding molecules comprising an antigen binding domain with a HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, an antigen-binding molecule comprises a HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 1. In some embodiments, an antigen-binding molecule comprises a HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 1, wherein the differences in the amino acid residue(s) relative to the sequence disclosed in Table 1 are conservative substitutions or moderately conservative substitutions. Antigen-Binding Proteins Comprising Fc Modifications

[0323] In some embodiments, an antigen-binding molecule as disclosed herein (e.g., a BCMAxCD3 bispecific antigen-binding molecule such as an anti-BCMAxCD3 bispecific antibody or a CD20xCD3 bispecific antigen-binding molecule such as an anti-CD20xCD3 bispecific antibody) comprises an Fc domain comprising one or more modifications or mutations that enhance or diminish antibody binding to the FcRn receptor. For example, the present disclosure includes antigen-binding molecules comprising one or more mutations in the CH2 and / or CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.

[0324] Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., EorQ); 250 and 428 (e.g., Lor F); 252 (e.g., L / Y / F / WorT), 254 (e.g., SorT), and 256 (e.g., S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). See, e.g.., Ko et al., BioDrugs 2021, 35:147-157.

[0325] In certain embodiments, a BCMAxCD3 bispecific antigen-binding molecule or a CD20xCD3 bispecific antigen-binding molecule comprises an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F).

[0326] In some embodiments, the BCMAxCD3 bispecific antigen-binding molecules or the CD20xCD3 bispecific antigen-binding molecules of the present disclosure comprise a modified Fc domain having reduced effector function. As used herein, a "modified Fc domain having reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc., relative to a wild-type, naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis and opsonization, relative to a comparator molecule comprising the wild-type, naturally occurring version of the Fc portion. In certain embodiments, a "modified Fc domain having reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcyR).

[0327] In certain embodiments, a modified Fc domain having reduced binding to an Fc receptor (e.g., Fey receptor, e.g., FcyRI, FcyRIIA, FcyRIIB, or FcyRHIA) is a variant lgG1 Fc ora variant lgG4 Fc comprising one or more substitutions or modifications in the hinge region and / or a CH region (e.g., CH2). For example, a modified Fc domain may comprise a variant lgG1 Fc wherein at least one amino acid of an IgG 1 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an lgG2 Fc hinge region and / or CH region. In certain embodiments, the modified Fc domain is a variant IgG 1 Fc or a variant lgG4 Fc comprising one or more substitutions or modifications in the hinge region. For example, a modified Fc domain may comprise a variant lgG1 Fc, wherein at least one amino acid of the IgG 1 Fc hinge region is replaced with the corresponding amino acid from the lgG2 Fc hinge region. In one example, the variant IgG 1 Fc can comprise a human lgG2 lower hinge amino acid sequence or can comprise both a human lgG2 lower hinge amino acid sequence and a human lgG4 CH2 amino acid sequence. For example, in some embodiments, the heavy chain constant region can comprise a variant lgG1 Fc in which positions 233-236 by EU numbering are occupied by PVA. See, e.g., US 10,988,537, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the heavy chain constant region can comprise a variant IgG 1 Fc in which the IgG 1 CH2 region is replaced with the corresponding amino acids from the lgG4 CH2 region and in which positions 233-236 by EU numbering are occupied by PVA. Alternatively, a modified Fc domain may comprise a variant lgG4 Fc wherein at least one amino acid of an lgG4 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an lgG2 Fc hinge region and / or CH region. Alternatively, a modified Fc domain may comprise a variant lgG4 Fc, wherein at least one amino acid of the lgG4 Fc hinge region is replaced with the corresponding amino acid from the lgG2 Fc hinge region. In one example, the variant lgG4 Fc can comprise a human lgG2 lower hinge amino acid sequence. For example, in some embodiments, the heavy chain constant region can comprise a variant lgG4 Fc in which positions 233-236 by EU numbering are occupied by PVA. See, e.g., US 10,988,537, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, a modified Fc domain comprises a modified hinge region in which each of positions 233-236 by EU numbering is occupied by G or is unoccupied. In some embodiments, a modified Fc domain comprises modifications in which each of positions 233-236 by EU numbering is occupied by G or is unoccupied. For example, in some embodiments, a modified Fc domain can comprise a modified hinge region in which positions 233-236 by EU numbering are occupied by GGG. See, e.g., US 11,518,807, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the heavy chain constant region can comprise a variant IgG 1 Fc in which the lgG1 CH2 region is replaced with the corresponding amino acids from the lgG4 CH2 region and in which positions 233-236 by EU numbering are occupied by GGG. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent No. 11,518,807, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions set forth therein. Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications as set forth in US 8,697,396, US 10,988,537, US 2014 / 0171623, US 2014 / 0134162, US 2014 / 0243504, and WO 2014 / 043361, the disclosures of each of which are incorporated by reference herein.

[0328] All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure. Polynucleotides, Vectors, and Host Cells

[0329] In another aspect, the present disclosure provides nucleic acid molecules comprising one or more polynucleotide sequences encoding the antigen-binding molecules disclosed herein, as well as vectors (e.g., expression vectors) encoding such polynucleotide sequences and host cells into which such vectors have been introduced.

[0330] Polynucleotides, as disclosed herein, may encode all or a portion of an antigen-binding molecule, antibody, or antigen-binding fragment as disclosed throughout the present disclosure. In some cases, a single polynucleotide may encode both a HCVR and a LCVR (e.g., defined with reference to the CDRs contained within the respective amino acid sequence-defined HCVR and LCVR, defined with reference to the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined with reference to the amino acid sequences of the HCVR and LCVR, respectively) of an antibody or antigen-binding fragment, or the HCVR and LCVR may be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case, in which the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined in a single vector or may be contained in separate vectors (i.e., a pair of vectors). In any case, a host cell used to express the polynucleotide(s) or vector(s) may contain the full complement of component parts to generate the antibody or antigen-binding fragment thereof. For example, a host cell may comprise separate vectors, each encoding a HCVR and a LCVR, respectively, of an antibody or antigen-binding fragment thereof as discussed above or herein. Similarly, the polynucleotide or polynucleotides, and the vector or vectors, may be used to express the full-length heavy chain and full-length light chain of an antibody as discussed above or herein. For example, a host cell may comprise a single vector with polynucleotides encoding both a heavy chain and a light chain of an antibody, or the host cell may comprise separate vectors with polynucleotides encoding, respectively, a heavy chain and a light chain of an antibody as disclosed above or herein.

[0331] In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding an antigen-binding molecule disclosed in Table 1.

[0332] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-BCMA HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4, 6, and 8, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-BCMA HCVR comprising or consisting of the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 1, ora polynucleotide sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to SEQ ID NO: 1.

[0333] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 28, 30, and 32, respectively; or of SEQ ID NOs: 36, 38, and 40, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising or consisting of the sequence of SEQ ID NO: 26 or 34. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 25 or 33, or a polynucleotide sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to SEQ ID NO: 25 or 33.

[0334] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 20, 22, and 24, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising or consisting of the sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO: 17, or a polynucleotide sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to SEQ ID NO: 17.

[0335] In some embodiments, compositions are provided comprising one or more nucleic acid molecules as disclosed herein. For example, in some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a first antigen-binding domain that binds BCMA, and a second nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a second antigenbinding domain that binds CD3. In some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a first antigenbinding domain that binds BCMA, a second nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a first antigen-binding domain that binds BCMA, a third nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a second antigenbinding domain that binds CD3, and a fourth nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a second antigen-binding domain that binds CD3. In some embodiments, an anti-BCMA HCVR comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4, 6, and 8, respectively. In some embodiments, an anti-BCMA LCVR comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 20, 22, and 24, respectively. In some embodiments, an anti-CD3 HCVR comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 28, 30, and 32, respectively; or the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 36, 38, and 40, respectively. In some embodiments, an anti-CD3 LCVR comprises the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 20, 22, and 24, respectively.

[0336] In one embodiment, the present disclosure provides a nucleic acid molecule or nucleic acid molecules that comprise a nucleotide sequence encoding the HCVR sequence of the anti-BCMA antigen-binding domain comprising SEQ ID NO: 2, a nucleotide sequence encoding the HCVR sequence of the anti-CD3 antigen-binding domain comprising SEQ ID NO: 26, and a nucleotide sequence encoding the LCVR sequence comprising SEQ ID NO: 18.

[0337] In one embodiment, the present disclosure provides a nucleic acid molecule or nucleic acid molecules that comprise a nucleotide sequence encoding the HCVR sequence of the anti-BCMA antigen-binding domain comprising SEQ ID NO: 2, a nucleotide sequence encoding the HCVR sequence of the anti-CD3 antigen-binding domain comprising SEQ ID NO: 34, and a nucleotide sequence encoding the LCVR sequence comprising SEQ ID NO: 18.

[0338] In another aspect, the present disclosure also provides recombinant expression vectors carrying one or more nucleic acid molecules as disclosed herein, as well as host cells into which such vectors have been introduced. In some embodiments, the host cell is a prokaryotic cell (e.g., E. coli). In some embodiments, the host cell is a eukaryotic cell, such as a non-human mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell). Also provided herein are methods of producing the antigen-binding molecules of the disclosure by culturing the host cells under conditions permitting production of the antigen-binding molecules, and recovering the antigen-binding molecules so produced.

[0339] In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding an antigen-binding molecule disclosed in Table 2.

[0340] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD20 HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 47, 48, and 49, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD20 HCVR comprising or consisting of the sequence of SEQ ID NO: 44.

[0341] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 53, 54, and 55, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising or consisting of the sequence of SEQ ID NO: 46.

[0342] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 50, 51, and 52, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising or consisting of the sequence of SEQ ID NO: 45.

[0343] In some embodiments, compositions are provided comprising one or more nucleic acid molecules as disclosed herein. For example, in some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a first antigen-binding domain that binds CD20, and a second nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a second antigenbinding domain that binds CD3. In some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a first antigenbinding domain that binds CD20, a second nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a first antigen-binding domain that binds CD20, a third nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a second antigenbinding domain that binds CD3, and a fourth nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a second antigen-binding domain that binds CD3. In some embodiments, an anti-CD20 HCVR comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 47, 48, and 49, respectively. In some embodiments, an anti-CD20 LCVR comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 50, 51, and 52, respectively. In some embodiments, an anti-CD3 HCVR comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 53, 54, and 55, respectively. In some embodiments, an anti-CD3 LCVR comprises the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 50, 51, and 52, respectively.

[0344] In one embodiment, the present disclosure provides a nucleic acid molecule or nucleic acid molecules that comprise a nucleotide sequence encoding the HCVR sequence of the anti-CD20 antigen-binding domain comprising SEQ ID NO: 44, a nucleotide sequence encoding the HCVR sequence of the anti-CD3 antigen-binding domain comprising SEQ ID NO: 46, and a nucleotide sequence encoding the LCVR sequence comprising SEQ ID NO: 45.

[0345] In another aspect, the present disclosure also provides recombinant expression vectors carrying one or more nucleic acid molecules as disclosed herein, as well as host cells into which such vectors have been introduced. In some embodiments, the host cell is a prokaryotic cell (e.g., E. coli). In some embodiments, the host cell is a eukaryotic cell, such as a non-human mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell). Also provided herein are methods of producing the antigen-binding molecules of the disclosure by culturing the host cells under conditions permitting production of the antigen-binding molecules, and recovering the antigen-binding molecules so produced. Characterization of BCMAxCD3 Bispecific Antigen-Binding Molecules

[0346] The present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies) and functional fragments thereof that bind to BCMA and CD3 (e.g., human BCMA and CD3) with high affinity.

[0347] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein) that bind BCMA and CD3 (e.g., at 25°C or 37°C) with a Kd of less than about 75 nM, e.g., as measured by surface plasmon resonance or a substantially similar assay. In certain embodiments, the antigen-binding molecules of the present disclosure bind human BCMA and CD3 with a KD of less than about 75 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured by surface plasmon resonance or a substantially similar assay.

[0348] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein) that specifically interact (e.g., bind with) cells that express BCMA and / or CD3. The extent to which an antigen-binding molecule binds cells that express BCMA and / or CD3 can be assessed by flow cytometry. For example, in some embodiments, the present disclosure provides anti-BCMAxCD3 bispecific antibodies that specifically bind cells that express BCMA and / or CD3 on the cell surface (e.g., human plasma cells and / or T cells). In some embodiments, the disclosure provides anti-BCMAxCD3 bispecific antibodies that bind BCMA and / or CD3-expressing cells or cell lines with an ECso value of about 10 nM or less, e.g., from about 0.5 nM to about 10 nM, e.g., an ECso value of about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, or about 10 nM, e.g., as determined by flow cytometry or a substantially similar assay. Characterization of CD20xCD3 Bispecific Antigen-Binding Molecules

[0349] The present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies) and functional fragments thereof that bind to CD20 and CD3 (e.g., human CD20 and CD3) with high affinity.

[0350] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein) that specifically interact (e.g., bind with) cells that express CD20 and / or CD3. The extent to which an antigen-binding molecule binds cells that express CD20 and / or CD3 can be assessed by an in vitro binding assay. For example, in some embodiments, the present disclosure provides anti-CD20xCD3 bispecific antibodies that specifically bind cells that express CD20 and / or CD3 on the cell surface (e.g., human B cells and / or T cells). In certain embodiments, the anti-CD20*CD3 bispecific antibodies bind Jurkat cells and Raji cells with an EC50 value of less than about 60 nM, as measured by an in vitro binding assay. In certain embodiments, the anti-CD20*CD3 bispecific antibodies bind CD3 or CD20 on the surface of a Jurkat or Raji cell, respectively, with an EC50 value of less than about 1000 mM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 70 nM, less than about 65 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 100 pM, less than about 10 pM, or less than about 1 pM as measured by an in vitro binding assay. Epitope Mapping and Related Technologies

[0351] In some embodiments, the epitope on BCMA and / or CD20 and / or CD3 to which the antigen-binding molecules of the present disclosure bind (e.g., an epitope of BCMA or CD20 to which a first antigen-binding domain (D1) binds, or an epitope of CD3 to which a second antigen-binding domain (D2) binds) may consist of a single contiguous sequence of 3 or more (e.g., 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a BCMA or CD20 or CD3 protein. Alternatively, the epitope may consist of a plurality of noncontiguous amino acids (or amino acid sequences) of a BCMA or CD20 or CD3 protein. The antibodies of the invention may interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or may interact with amino acids on two or more different CD3 chains. The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0352] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques that can be used to determine an epitope or binding domain of a particular antibody or antigen-binding domain include, e.g., routine crossblocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:25GA-2Q5A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antibody interacts.

[0353] The present disclosure also includes antigen-binding molecules (e.g., antibodies or antigen-binding domains thereof) that bind to the same epitope as, or competes for binding with, a bispecific BCMAxCD3 antigen-binding molecule or a bispecific CD20xCD3 antigen-binding molecule described herein. One skilled in the art can determine whether or not a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present disclosure by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on BCMA and / or CD20 and / or CD3 as a reference bispecific antigen-binding molecule of the present disclosure, the reference bispecific molecule is first allowed to bind to a BCMA and / or CD20 and / or CD3 protein. Next, the ability of a test antibody to bind to the BCMA and / or CD20 and / or CD3 molecule is assessed. If the test antibody is able to bind to BCMA and / or CD20 and / or CD3 following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of BCMA and / or CD20 and / or CD3 than the reference bispecific antigenbinding molecule. On the other hand, if the test antibody is not able to bind to the BCMA and / or CD20 and / or CD3 molecule following saturation binding with the reference bispecific antigenbinding molecule, then the test antibody may bind to the same epitope of BCMA and / or CD20 and / or CD3 as the epitope bound by the reference bispecific antigen-binding molecule of the disclosure. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, radioimmunoassay (RIA), Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present disclosure, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 2-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigenbinding proteins are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[0354] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a BCMA and / or CD20 and / or CD3 protein under saturating conditions followed by assessment of binding of the test antibody to the BCMA and / or CD20 and / or CD3 molecule. In a second orientation, the test antibody is allowed to bind to a BCMA and / or CD20 and / or CD3 molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the BCMA and / or CD20 and / or CD3 molecule. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the BCMA and / or CD20 and / or CD3 molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to BCMA and / or CD20 and / or CD3. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope. Preparation of Antigen-Binding Domains and Construction of Multispecific AntigenBinding Molecules

[0355] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigen-binding domains can be appropriately arranged relative to one another to produce a bispecific antigenbinding molecule of the present disclosure using routine methods. A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present disclosure is provided elsewhere herein. In certain embodiments, one or more of the individual components (e.g., heavy, and light chains) of the multispecific antigenbinding molecules are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the present disclosure can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., BCMA or CD20 or CD3) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules.

[0356] In some embodiments, genetically engineered animals may be used to make human bispecific antigen binding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011 / 0195454, the entire contents of which are incorporated herein by reference, for a detailed discussion of such engineered mice and the use thereof to produce bispecific antigen-binding molecules). As used herein, "fully human" refers to an antigen-binding molecule, e.g., an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antigen-binding molecule, antibody, antigen-binding fragment, or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In other instances, the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g., compared to any wild-type human immunoglobulin regions or domains. Bioequivalents

[0357] The present disclosure encompasses antigen-binding molecules having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind BCMA and / or CD20 and / or CD3. Such variant molecules comprise one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antigen-binding molecules. Likewise, the nucleic acid sequences encoding the antigen-binding molecules of the present disclosure encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antigen binding molecule that is essentially bioequivalent to the antigen-binding molecules disclosed herein.

[0358] The present disclosure includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins, e.g., bispecific antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0359] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0360] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the first antigen-binding protein (e.g., reference product) and the second antigen-binding protein (e.g., biological product) without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0361] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0362] Bioequivalence may be demonstrated by in vivo and in vitro methods. Non-limiting examples of bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0363] Bioequivalent variants of the exemplary bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other embodiments, bioequivalent antibodies may include the exemplary bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the antibodies, e.g., mutations which eliminate or remove glycosylation. Immunoglobulin Depleting Agents

[0364] In various aspects, the present disclosure provides immunoglobulin depleting agents, e.g., which may be combined with or administered in combination with plasma cell depleting agents (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof) or B cell depleting agents (e.g., an anti-CD20xCD3 bispecific antibody, or a functional fragment thereof) described herein. In some embodiments, the immunoglobulin depleting agent may be administered in combination with a plasma cell depleting agent, a B cell depleting agent, plasmapheresis, therapeutic plasma exchange, immunoadsorption, and / or an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein. In some embodiments, an immunoglobulin depleting agent may be useful for, e.g., accelerating IgG clearance. In some embodiments, an immunoglobulin depleting agent is capable of accelerating IgG serum clearance.

[0365] In some embodiments, an immunoglobulin depleting agent may comprise a neonatal Fc receptor (FcRn) blocker such as, but not limited to, efgartigimod alfa. The mechanistic concept of FcRn-targeting therapeutics is to accelerate IgG catabolism by blocking the FcRn-mediated intracellular IgG recycling pathway, thereby reducing overall plasma IgG levels. FcRn can participate in the maintenance of IgG levels by salvaging IgG from lysosomal degradation, thereby prolonging the half-life of IgG. In some embodiments, FcRn blockers can compete with IgG for binding to FcRn. Due to their higher affinity for FcRn, FcRn blockers can prevent IgG from binding to FcRn and, instead, IgG is transported to the lysosome and degraded, thereby leading to decreased circulating levels of IgG.

[0366] In some embodiments, an FcRn blocker can include Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), Nipocalimab (M281), Orilanolimab (SYNT001), IMVT-1402, or any combination thereof. See, e.g., Zuercheret al. (2019) Autoimmun. Rev. 18(10):102366, which is incorporated herein by reference in its entirety.

[0367] In some embodiments, an immunoglobulin depleting agent may comprise an IgG degrading enzyme such as IdeS (imlifidase), IdeZ, or IdeXork. IdeS (imlifidase) is an endopeptidase derived from Streptococcus pyogenes which has specificity for human IgG, and when infused intravenously results in rapid cleavage of IgG. IdeZ (immunoglobulin-degrading enzyme from Streptococcus equi subspecies zooepidemicus) is an engineered recombinant protease overexpressed in Escherichia coli. IdeZ specifically cleaves IgG molecules below the hinge region to yield F(ab')2 and Fc fragments. IdeXork (Xork) is yet another example of an IgG protease. More particular non-limiting examples of IgG degrading enzymes include Imlifidase / IdeS / Fabricator, IdeZ, IceM, IceMG, CYR-212, CYR-241, S-1117, HNSA-5487, and Xork. In some embodiments, an immunoglobulin depleting agent may facilitate IgG degradation via lysosomal destruction. A non-limiting example of an immunoglobulin depleting agent which may facilitate IgG degradation via lysosomal destruction is BHV-1300. Plasmapheresis, Therapeutic Plasma Exchange, and Immunoadsorption

[0368] In various aspects, the methods disclosed herein can include plasmapheresis, therapeutic plasma exchange, or immunoadsorption. These can be combined, for example, with treatment with plasma cell depleting agents (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof), B cell depleting agents (e.g., an anti-CD20xCD3 bispecific antibody, or a functional fragment thereof), and / or immunoglobulin depleting agents described herein. In some embodiments, the plasmapheresis, therapeutic plasma exchange, or immunoadsorption may be performed in combination with treatment with a plasma cell depleting agent, a B cell depleting agent, and / or an immunogen (e.g., an immunogenic delivery vehicle) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein. Plasmapheresis, therapeutic plasma exchange, and immunoadsorption may be useful strategies for removal of AAV antibodies from patients’ blood plasma.

[0369] Plasmapheresis is a process used to selectively remove blood components used to treat a variety of conditions including those caused by the acute overproduction of antibodies (e.g., autoimmunity, transplant rejection), in which removal of pathogenic immunoglobulins results in clinical benefit. Immunoadsorption is a selective therapeutic apheresis technique by which immunoglobulins are selectively removed from patients’ plasma. The immunoadsorption can be, for example, total immunoglobulin immunoadsorption. See, e.g., Boedecker-Lips et al. (2023) J. Clin. Apher. 38(5):590-601. Alternatively, the immunoadsorption can be AAV capsid specific immunoadsorption. See, e.g., Bertin et al. (2020) Sci. Rep. 10:864. Combinations Comprising a Plasma Cell Depleting Agent

[0370] A plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) can be administered to a subject in need thereof either alone, or in combination with, a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule), an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod), and / or an immunogen. In some embodiments, the administration of the plasma cell depleting, the B cell depleting agent, the immunoglobulin depleting agent, and / or the immunogen can be further combined with plasmapheresis, therapeutic plasma exchange, and / or immunoadsorption. As used herein, the term “in combination with”, e.g., a BCMAxCD3 bispecific antigen-binding molecule (or other immunomodulator or immunogen, etc.) means that additional component(s) may be administered prior to, concurrent with, or after the administration of BCMAxCD3 bispecific antigen-binding molecule (or other immunomodulator or immunogen, etc.) molecule (or other immunomodulator or immunogen, etc.). The different components of the combination can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component, for example, wherein the further agent is in a separate formulation).

[0371] For example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) can be administered to a subject in need thereof either alone, or in combination with, a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule) and / or an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod). In some embodiments, the B cell depleting agent is administered before, at the same time as, or after the plasma cell depleting agent. In some embodiments, the immunoglobulin depleting agent is administered after the plasma cell depleting agent.

[0372] In one example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule).

[0373] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod). In some embodiments, the immunoglobulin depleting agent comprises an FcRn blocker. In some embodiments, the immunoglobulin depleting agent comprises an IgG degrading enzyme.

[0374] In some embodiments, the combination of the plasma cell depleting agent and the immunoglobulin depleting agent, when administered in further combination with an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) to a subject in need thereof, decreases a level of an anti-immunogen antibody titer (e.g., an anti-AAV antibody titer) in the subject (e.g., such as can be measured in a serum sample isolated from the subject). In some embodiments, the level of the anti-immunogen antibody titer is decreased by about 1-fold to about 20-fold, about 2-fold to about 15-fold, about 4-fold to about 10-fold, about 3-fold to about 18-fold, about 5-fold to about 12-fold, or about 6-fold to about 8-fold, as compared to the level of the anti-immunogen antibody titer in a subject administered the immunogen alone. In some embodiments, the anti-immunogen antibody titer is decreased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold, or more. In some embodiments, the anti-immunogen antibody titer is decreased by about 20-fold.

[0375] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule) and an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod). In some embodiments, the immunoglobulin depleting agent comprises an FcRn blocker. In some embodiments, the immunoglobulin depleting agent comprises an IgG degrading enzyme.

[0376] In some embodiments, the combination of the plasma cell-depleting agent, the B cell depleting agent, and the immunoglobulin-depleting agent, when administered in further combination with an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) to a subject in need thereof, decreases the level of an anti-immunogen antibody titer (e.g., an anti-AAV antibody titer) in the subject (e.g., such as can be measured in a serum sample isolated from the subject). In some embodiments, the level of the anti-immunogen antibody titer may be decreased by about 1-fold to about 20-fold, about 2-fold to about 15-fold, about 4-fold to about 10-fold, about 3-fold to about 18-fold, about 5-fold to about 12-fold, about 6-fold to about 8-fold, about 10-fold to about 30-fold, about 20-fold to about 50-fold, about 30-fold to about 70-fold, about 40-fold to about 90-fold, or about 50-fold to about 100-fold, as compared to the level of the anti-immunogen antibody titer in a subject administered the immunogen alone. In some embodiments, the anti-immunogen antibody titer is decreased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold, or more. In some embodiments, the anti-immunogen antibody titer is decreased by about 100-fold.

[0377] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with plasmapheresis, therapeutic plasma exchange, or immunoadsorption.

[0378] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with plasmapheresis, therapeutic plasma exchange, or immunoadsorption and a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule).

[0379] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with plasmapheresis, therapeutic plasma exchange, or immunoadsorption and an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod). In some embodiments, the immunoglobulin depleting agent comprises an FcRn blocker. In some embodiments, the immunoglobulin depleting agent comprises an IgG degrading enzyme.

[0380] In another example, a plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with plasmapheresis, therapeutic plasma exchange, or immunoadsorption, a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule), and an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod). In some embodiments, the immunoglobulin depleting agent comprises an FcRn blocker. In some embodiments, the immunoglobulin depleting agent comprises an IgG degrading enzyme.

[0381] In some embodiments, the B cell depleting agent comprises two or more B cell depleting agents (e.g., an anti-CD19 antigen-binding molecule and an anti-CD20 antigenbinding molecule). In some embodiments, the immunoglobulin depleting agent comprises two or more immunoglobulin depleting agents (e.g., an FcRn blocker and an IgG degrading enzyme).

[0382] In embodiments in which a plasma cell depleting agent (e.g., a BCMAxCD3 antigenbinding molecule) is administered to a subject in combination with, a B cell depleting agent (e.g., a CD20xCD3 antigen-binding molecule) and / or an immunoglobulin depleting agent (e.g., an FcRn blocker, such as Efgartigimod) and / or plasmapheresis, therapeutic plasma exchange, or immunoadsorption, one or more or all treatments can occur together or one or more or all treatments can occur sequentially. For example, in some embodiments in which the plasma cell depleting agent (e.g., a BCMAxCD3 antigen-binding molecule) is administered to a subject in need thereof in combination with an IgG degrading enzyme, the plasma cell depleting agent can be administered to the subject first, followed by the IgG degrading enzyme. In another example, in embodiments where an immunoglobulin depleting agent (e.g., FcRn blocker) is administered together with plasmapheresis, therapeutic plasma exchange, or immunoadsorption, the plasmapheresis, therapeutic plasma exchange, or immunoadsorption can be first followed by administration of the immunoglobulin depleting agent (e.g., FcRn blocker). Pharmaceutical Compositions

[0383] In another aspect, the present disclosure provides pharmaceutical compositions comprising plasma cell depleting agents (e.g., long-lived plasma cell (LLPC) depleting agents such as anti-BCMAxCD3 bispecific antibodies, or functional fragments thereof), B cell depleting agents (e.g., anti-CD19 and anti-CD20 antibodies, ora CD20xCD3 antigen-binding molecule (e.g., REGN1979), or functional fragments thereof), immunoglobulin depleting agents (e.g., neonatal Fc receptor (FcRn) blockers), and / or immunogens (e.g., immunogenic delivery vehicles) disclosed herein, optionally comprising a pharmaceutically acceptable carrier and / or excipient. In one specific embodiment, a composition described herein comprises an immunogen and an anti-CD20xCD3 bispecific antibody, or a functional fragment thereof, and optionally, further comprises a pharmaceutically acceptable carrier and / or excipient. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein.

[0384] The pharmaceutical compositions are formulated with one or more pharmaceutically acceptable vehicle, carriers, and / or excipients. Various pharmaceutically acceptable carriers and excipients are well-known in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0385] One exemplary embodiment of the present disclosure comprises a pharmaceutical composition comprising (i) a plasma cell depleting agent, (ii) a B cell depleting agent and / or an immunoglobulin depleting agent, and (iii) a pharmaceutically acceptable carrier and / or excipient. Another exemplary embodiment of the present disclosure comprises a pharmaceutical composition comprising (i) an immunogen, (ii) a plasma cell depleting agent, (iii) optionally, a B cell depleting agent and / or an immunoglobulin depleting agent, and (iv) a pharmaceutically acceptable carrier and / or excipient

[0386] In some embodiments, the plasma cell depleting agent comprises an antigen-binding molecule that specifically binds B cell maturation antigen (BCMA) and CD3. In some embodiments, the plasma cell depleting agent comprises an anti-BCMAxCD3 bispecific antibody, or functional fragment thereof, disclosed herein. Non-limiting examples of an anti-BCMAxCD3 bispecific antibody include linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

[0387] In some embodiments, the anti-BCMAxCD3 bispecific antibody comprises: (a) a first antigen-binding domain (D1) that binds an epitope of human BCMA; and (b) a second antigen-binding domain (D2) that binds an epitope of human CD3.

[0388] In some embodiments, the B cell depleting agent comprises anti-CD19 and anti-CD20 antibodies, or functional fragments thereof, disclosed herein. In some embodiments, the B cell depleting agent comprises a CD20xCD3 antigen-binding molecule (e.g., REGN1979).

[0389] In some embodiments, the immunoglobulin depleting agent comprises a neonatal Fc receptor (FcRn) blocker. A non-limiting example of an FcRn blocker is efgartigimod alfa. In some embodiments, the immunoglobulin depleting agent comprises an IgG degrading enzyme.

[0390] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid. In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle. In some embodiments, the immunogen is an immunogenic delivery vehicle and / or transgene product(s).

[0391] In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, a protozoal vector, or a mammalian cell.

[0392] In some embodiments, the immunogenic delivery vehicle is a viral vector.

[0393] In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, a retrovirus, or an oncolytic virus.

[0394] In some embodiments, the viral vector is AAV. In some embodiments, the viral vector is derived from AAV.

[0395] In some embodiments, the retrovirus is a lentivirus.

[0396] In some embodiments, the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.

[0397] In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intratumoral, intrathecal, transdermal, topical, or subcutaneous administration.

[0398] In some embodiments, the pharmaceutical composition comprises an injectable preparation, such as a dosage form for intravenous, subcutaneous, intracutaneous, and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described above, in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule.

[0399] The dose of a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen (e.g., immunogenic delivery vehicle) administered to a patient according to the present disclosure may vary depending upon the age and the size of the patient, symptoms, conditions, route of administration, and the like. The dose is typically calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering pharmaceutical compositions as disclosed herein may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0400] In some embodiments, e.g., for methods and compositions of the present disclosure involving administration of a plasma cell depleting agent which is a bispecific BCMAxCD3 antibody (e.g., REGN5458) to a subject, the dose of the bispecific BCMAxCD3 antibody (or pharmaceutical compositions thereof) is from about 1 mg / kg to about 30 mg / kg, such as from about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 15 mg / kg, about 15 mg / kg to about 20 mg / kg, about 20 mg / kg to about 25 mg / kg, or about 25 mg / kg to about 30 mg / kg. In some embodiments, the bispecific BCMAxCD3 antibody (e.g., REGN5458) can be administered to the subject at a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, or about 30 mg / kg. In one specific embodiment, the bispecific BCMAxCD3 antibody (e.g., REGN5458) (or pharmaceutical composition thereof) dose is about 20 mg / kg.

[0401] In some embodiments, e.g., for methods and compositions of the present disclosure involving administration of a B cell depleting agent which is a bispecific CD20xCD3 antibody (e.g., REGN1979) to a subject, the dose of the bispecific CD20xCD3 antibody (or pharmaceutical compositions thereof) is from about 0.05 mg / kg to about 3 mg / kg, such as from about 0.05 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2 mg / kg, about 2 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 3 mg / kg. In one specific embodiment, the bispecific CD20xCD3 antibody (e.g., REGN1979) is administered to the subject at a dose of about 0.1 mg / kg. In another specific embodiment, the bispecific CD20xCD3 antibody (e.g., REGN1979) is administered to the subject at a dose of about 1 mg / kg.

[0402] In some embodiments, e.g., for methods and compositions of the present disclosure involving administration of an immunoglobulin depleting agent which is efgartigimod to a subject, the dose of efgartigimod (or pharmaceutical compositions thereof) is from about 1 mg / kg to about 30 mg / kg, such as from about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 15 mg / kg, about 15 mg / kg to about 20 mg / kg, about 20 mg / kg to about 25 mg / kg, or about 25 mg / kg to about 30 mg / kg. In some embodiments, efgartigimod can be administered to the subject at a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, or about 30 mg / kg. In one specific embodiment, the efgartigimod (or pharmaceutical composition thereof) dose is about 20 mg / kg.

[0403] In some embodiments, e.g., for methods and compositions of the present disclosure involving administration of an immunogen which is an AAV to a subject, the dose of the AAV (or pharmaceutical compositions thereof) administered to a subject is between about 1x105 plaque forming units (pfu) to about 1x1015 pfu. In some cases, the AAV can be administered to the subject at a dose from about 1x108 pfu to about 1x1015 pfu, or from about 1x1010 pfu to about 1x1015 pfu, or from about 1x108 pfu to about 1x1012 pfu.

[0404] In some embodiments, the dose of the AAV (or pharmaceutical compositions thereof) administered to the subject is between about 1x105 vg to about 1x1016vg. In certain embodiments, the dose of the AAV administered to the subject is between about 1x106 vg to about 1x109vg, about 1x107 vg to about 1x1010vg, about 1x108 vg to about 1x1011 vg, about 1x109 vg to about 1x1012 vg, about 1x1010 vg to about 1x1013vg, about 1x1011 vg to about 1x1014vg, about 1x1012 vg to about 1x1015vg, about 1x1013 vg to about 1x1016vg, or about 1x1014 vg to about 1x1016vg. In certain embodiments, the dose of the AAV administered to the subject is between about 1x101° vg to about 1x1016vg. In certain embodiments, the dose of the AAV administered to the subject is at least about 1x106 vg, at least about 1x107 vg, at least about 1x108 vg, at least about 1x109 vg, at least about 1x101° vg, at least about 1x1011 vg, at least about 1x1012 vg, at least about 1x1012 vg, at least about 1x1013 vg, at least about 1x1014 vg, or at least about 1x1015 vg. In certain embodiments, the vg is total vector genome per subject.

[0405] In some embodiments, the dose of the AAV (or pharmaceutical compositions thereof) administered to the subject is about 1x1012, 1x1013, 1x1014, 1x1015, and 1x1016 vector genomes (vg) / mL. Further examples of doses of AAV include about 1x1012, about 1x1013, about 1x1014, about 1x1015, and about 1x1016 vector genomes (vg) / mL, or between about 1x1012 to about 1x1016, between about 1x1012 to about 1x1015, between about 1x1012 to about 1x1014, between about 1x1012 to about 1x1013, between about 1x1013 to about 1x1016, between about 1x1014 to about 1x1016, between about 1x1015 to about 1x1016, or between about 1x1013 to about 1x1015 vg / mL.

[0406] Other examples of doses of AAV (or pharmaceutical compositions thereof) include about 1x1012, about 1x1013, about 1x1014, about 1x1015, and about 1x1016 vector genomes (vg) / kg of body weight, or between about 1x1012 to about 1x1016, between about 1x1012 to about 1x1015, between about 1x1012 to about 1x1014, between about 1x1012 to about 1x1013, between about 1x1013to about 1x1016, between about 1x1014 to about 1x1016, between about 1x1015 to about 1x1016, or between about 1x1013 to about 1x1015 vg / kg of body weight.

[0407] In one example, the AAV dose (or pharmaceutical compositions thereof) is between about 1x1013 to about 1x1014 vg / mL or vg / kg. In another example, the AAV dose is between about 1x1012 to about 1x1013 vg / mL or vg / kg (e.g., between about 1x1012 to about 1x1013 vg / kg). In another example, the AAV dose is between about 1x1012 to about 1x1014 vg / mL or vg / kg (e.g., between about 1x1012 to about 1x1014 vg / kg).

[0408] In one specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 3x1011 vg / kg. In one specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 6x1011 vg / kg. In another specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 9x1011 vg / kg. In another specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 3x1012 vg / kg. In one specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 1x1013 vg / kg. In another specific embodiment, the AAV dose (or pharmaceutical composition thereof) is about 6x1013 vg / kg.

[0409] Various delivery systems are known and can be used to administer the pharmaceutical composition, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing, e.g., recombinant viruses comprising any components of the compositions disclosed herein, and a soluble carrier system that takes advantage of receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intratumoral, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. In some embodiments, a pharmaceutical composition as disclosed herein is administered intravenously. In some embodiments, a pharmaceutical composition as disclosed herein is administered subcutaneously. In some embodiments, a pharmaceutical composition as disclosed herein is administered intratumorally.

[0410] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen, ora pharmaceutical composition(s) thereof, is contained within a container. Thus, in another aspect, containers comprising an antigen-binding molecule and / or pharmaceutical composition as disclosed herein are provided. For example, in some embodiments, an antibody and / or pharmaceutical composition is contained within a container selected from the group consisting of a glass vial, a syringe, a pen delivery device, and an autoinjector.

[0411] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen, ora pharmaceutical composition(s) thereof, of the present disclosure is delivered, e.g., subcutaneously or intravenously, such as with a standard needle and syringe. In some embodiments, the syringe is a pre-filled syringe. In some embodiments, a pen delivery device or autoinjector is used to deliver a pharmaceutical composition of the present disclosure (e.g., for subcutaneous delivery). A pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0412] Examples of suitable pen and autoinjector delivery devices include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having applications, e.g., in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL).

[0413] In some embodiments, the pharmaceutical compositions of the present disclosure can be delivered using a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0414] In some embodiments, pharmaceutical compositions as described herein are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In some embodiments, the amount of the antigen-binding molecule contained in the dosage form is about 5 to about 1000 mg, e.g., from about 5 to about 500 mg, from about 5 to about 100 mg, or from about 10 to about 250 mg.

[0415] Plasma cell depleting agents, B cell depleting agents, immunoglobulin depleting agents, and / or immunogens, introduced into the subject or cell can be provided in compositions comprising a carrier, thereby increasing the stability of the introduced molecules, e.g., prolonging the period under given conditions of storage (e.g., -20°C, 4°C, or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo. Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules.

[0416] Various methods and compositions are provided herein to allow for introduction of a molecule (e.g., a nucleic acid or protein) into a cell or subject. Methods for introducing molecules into various cell types are known and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0417] Transfection protocols, as well as protocols for introducing molecules into cells, may vary. Non-limiting transfection methods include chemical-based transfection methods using liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74 (4):1590-4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: W. H. Freeman and Company, pp. 96-97); dendrimers; or cationic polymers such as DEAE-dextran or polyethylenimine. Nonchemical methods include electroporation, sonoporation, and optical transfection. Particle-based transfection can include the use of a gene gun or magnet-assisted transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods can also be used for transfection.

[0418] Introduction of nucleic acids or proteins into a cell can also be mediated by electroporation, by intracytoplasmic injection, by viral infection, by adenovirus, by adeno-associated virus, by lentivirus, by retrovirus, by transfection, by lipid-mediated transfection, or by nucleofection. Nucleofection is an improved electroporation technology that enables nucleic acid substrates to be delivered not only to the cytoplasm, but also through the nuclear membrane and into the nucleus. In addition, use of nucleofection in the methods disclosed herein typically requires much fewer cells than regular electroporation (e.g., only about 2 million cells as compared with 7 million cells by regular electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[0419] Introduction of molecules (e.g., nucleic acids or proteins) into a cell (e.g., a zygote) can also be accomplished by microinjection. In zygotes (i.e., one-cell stage embryos), microinjection can be into the maternal and / or paternal pronucleus or into the cytoplasm. If the microinjection is into only one pronucleus, the paternal pronucleus is preferable due to its larger size.

[0420] Other methods for introducing molecules (e.g., nucleic acid or proteins) into a cell or subject can include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or implantable-device-mediated delivery. As specific examples, a nucleic acid or protein can be introduced into a cell or subject in a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule. Some specific examples of delivery to a subject include hydrodynamic delivery, virus-mediated delivery (e.g., adeno-associated virus (AAV)-mediated delivery), and lipid-nanoparticle-mediated delivery.

[0421] Introduction of nucleic acids or proteins into cells or subjects can be accomplished by hydrodynamic delivery (HDD). For gene delivery to parenchymal cells, only essential DNA sequences need to be injected via a selected blood vessel, eliminating safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is capable of reaching cells in the different tissues accessible to the blood. Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins, and other small compounds in vivo. See, e.g., Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, herein incorporated by reference in its entirety for all purposes.

[0422] Introduction of nucleic acids can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses / viral vectors which can be useful in accomplishing virus-mediated delivery include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome or, alternatively, do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and / or packaging). Viruses can cause transient expression or longer-lasting expression. Viral vectors may be genetically modified from their wild type counterparts. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some examples, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some examples, the viral vector may have an enhanced transduction efficiency. In some examples, the immune response induced by the virus in a host may be reduced. In some examples, viral genes (such as integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some examples, the viral vector may be replication defective. In some examples, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some examples, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell or population of host cells along with the vector system described herein. In other examples, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some examples, the vector system described herein may also encode the viral components required for virus amplification and packaging.

[0423] Exemplary viral titers (e.g., AAV titers) include about 1012, about 1013, about 1014, about 1015, and about 1016 vector genomes (vg) / mL, or between about 1012 to about 1016, between about 1012 to about 1015, between about 1012 to about 1014, between about 1012 to about 1013, between about 1013 to about 1016, between about 1014 to about 1016, between about 1015 to about 101S, or between about 1013 to about 1015 vg / mL. Other exemplary viral titers (e.g., AAV titers) include about 1012, about 1013, about 1014, about 1015, and about 1016 vector genomes (vg) / kg of body weight, or between about 1012 to about 1016, between about 1012 to about 1015, between about 1012 to about 1014, between about 1012 to about 1013, between about 1013 to about 1016, between about 1014 to about 1016, between about 1015 to about 1016, or between about 1013 to about 1015 vg / kg of body weight. In one example, the viral titer is between about 1013 to about 1014 vg / mL or vg / kg. In another example, the viral titer is between about 1012 to about 1013 vg / mL or vg / kg (e.g., between about 1012 to about 1013 vg / kg). In another example, the viral titer is between about 1012 to about 1014 vg / mL or vg / kg (e.g., between about 1012 to about 1014 vg / kg).

[0424] In yet another aspect, the present disclosure includes compositions and therapeutic formulations comprising any of the plasma cell depleting agents, B cell depleting agents, immunoglobulin depleting agents, and / or immunogens, described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof. In some embodiments, the additional therapeutic agent(s) is an immunomodulatory agent or anti-inflammatory agent. In some embodiments, the additional therapeutic agent(s) is immunosuppressive therapy. In some embodiments, the additional therapeutic agent(s) is a surgical procedure.

[0425] Exemplary additional therapeutic agents that may be combined with or administered in combination with any of the plasma cell depleting agents, B cell depleting agents, immunoglobulin depleting agents, and / or immunogens, of the present disclosure include, e.g., an anti-CD38 antibody (e.g., daratumumab), a proteasome inhibitor, a histone deacetylase inhibitor, a B-cell activating factor (BAFF) inhibitor, an APRIL inhibitor, a steroid (e.g., corticosteroids such as topical, systemic, oral, or inhaled corticosteroids including, but not limited to, betamethasone, clobetasol, dexamethasone, fluocinolone, fluocinonide, halobetasol, hydrocortisone, methylprednisolone, prednisone, prednisolone, or triamcinolone); a non steroidal topical medication such as, but not limited to, a phosphodiesterase 4 (PDE4) inhibitor or a calcineurin inhibitor; a non-steroidal anti-inflammatory drug (NSAID) such as, but not limited to, celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, rofecoxib, salicylates, sulfasalazinem, sulindac, ortolmetin; an anti-inflammatory antibody or biologic (e.g., an antitumor necrosis factor alpha (TNFa) antibody or biologic such as, but not limited to, adalimumab, certolizumab, etanercept, golimumab, or infliximab; an anti-l L1 antibody or biologic such as, but not limited to, LY2189102, anakinra, canakinumab, gerokizumab, or rilonacept; an antiinterleukin 6 (IL6) / IL-6 receptor (R) antibody or biologic such as, but not limited to, sarilumab, siltuximab, ortocilizumab; an anti-l L17A / IL-17R antibody or biologic such as, but not limited to, bimekizumab, brodalumab, ixekizumab, or secukinumab; or an anti-l L12 / IL-23 antibody or biologic such as, but not limited to, AMG139, BI655066, brazikumab, briankizumab, guselkumab, mirikizumab, risankizumab, tildrakizumab, or ustekinumab); a JAK inhibitor such as, but not limited to, abrocitinib, baricitinib, fedratinib, filgotinib, ruxolitinib, tofacitinib, or upadacitinib; an immunosuppressive agent (e.g., a systemic immunosuppressant such as, but not limited to, methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, mycophenolate mofetil, or azathioprine); a disease-modifying antirheumatic drug (DMARD) such as, but not limited to, apremilast, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, or tofacitinib; radiation therapy; chemotherapy; intravenous immunoglobulin therapy; or a surgery or a surgical procedure (such as, but not limited to, splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukapheresis, therapeutic plasma exchange, immunoadsorption, or cell, tissue, or organ transplantation). In some embodiments, the surgery or surgical procedure as described herein is used in combination with the anti-BCMAxCD3 bispecific antibody or the anti-CD20xCD3 bispecific antibody and in place of the FcRn blocker.

[0426] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen described herein may be administered with an additional therapeutic agent comprising, e.g., a broad-spectrum immunosuppression methodology, or combination thereof, including broad spectrum immunosuppression, e.g., calcineurin inhibitors (tacrolimus, cyclosporine), rapamycin, MMF, corticosteroids, methotrexate, proteasome inhibitors, costimulation blockade (CTLA4-lg / abatacept / belatacept), Src kinase inhibitors (dasatinib), Btk inhibitors (acalabrutinib), B cell depleting agents (rituximab), IgG degrading enzymes (IdeS), IgG half-life reducers (FcRn blockers), or combinations thereof.

[0427] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of the plasma cell depleting agent, the B cell depleting agent, the immunoglobulin depleting agent, and / or the immunogen, or the pharmaceutical composition(s) thereof, of the present disclosure. Such administration regimens can be considered, for example, the administration of a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen, ora pharmaceutical composition(s) thereof, “in combination with” an additional therapeutically active component.

[0428] The present disclosure includes pharmaceutical compositions in which a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen (e.g., an immunogenic delivery vehicle) of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

[0429] Therapeutic or pharmaceutical compositions comprising the compositions or combinations disclosed herein can be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. See a / so Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J. Pharm. Sc / . Technol. 52:238-311. In certain embodiments, the pharmaceutical compositions are non-pyrogenic. Methods of Use

[0430] In various aspects, the present disclosure provides methods for inhibiting or preventing an immune response to an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV) in a subject in need thereof, the methods comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent disclosed herein. In some embodiments, the present disclosure provides methods for inhibiting or preventing generation of antibodies (e.g., neutralizing antibodies) to an immunogen in a subject in need thereof, the methods comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent. In some embodiments of methods of the disclosure comprising administering to the subject an effective amount of a plasma cell depleting agent and an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV), the subject has a pre-existing immunity against the immunogen (e.g., AAV). In another aspect, provided herein is a method for inhibiting generation of neutralizing antibodies to an immunogen in a subject in need thereof (e.g., a subject without a pre-existing immunity against the immunogen), the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof. In some embodiments, the present disclosure provides methods for increasing effectiveness of re-administration of an immunogen to a subject in need thereof, the methods comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent In another aspect, provided herein is a method for increasing effectiveness of re-administration of an immunogen to a subject in need thereof (e.g., a subject without a pre-existing immunity against the immunogen), the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof. The term “re-administering” is used synonymously and interchangeably with the term “re-dosing” herein. In some embodiments, the present disclosure provides methods for increasing or maintaining the level of a transgene expression in a subject in need thereof, and the transgene is delivered to the subject via an immunogenic delivery vehicle (e.g., an AAV), the methods comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent.

[0431] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent which can be useful in any of the methods or compositions disclosed herein may be further used in combination with a plasmapheresis, therapeutic plasma exchange, and / or immunoadsorption.

[0432] In some embodiments, administration of a plasma cell depleting agent, a B cell depleting agent, an immunoglobulin depleting agent, and / or an immunogen prevents or delays the increase of disease symptoms or the progression of disease in a subject having disease or condition.

[0433] The term “immune response” refers to a response of a cell of the immune system (e.g., a B-cell, T-cell, macrophage or polymorphonucleocyte) to a stimulus such as an immunogen, e.g., an antigen (e.g., a viral antigen). Active immune responses can involve differentiation and proliferation of immunocompetent cells, which leads to synthesis of antibodies or the development of cell-mediated reactivity, or both. An active immune response can be mounted by the host after exposure to an antigen (e.g., by infection or by vaccination). An active immune response can be contrasted with passive immunity, which can be acquired through the transfer of substances such as, e.g., an antibody, a transfer factor, a thymic graft, and / or a cytokine from an actively immunized host to a non-immune host.

[0434] In some embodiments, the immune response is a humoral (antibody producing) immune response and / or a cell-mediated immune response in a subject (e g., a human).

[0435] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent may inhibit an immune response by a cell (e.g., an immune cell such as by a B cell or a T cell) or by an immune system of a subject (e.g., a human) which can be elicited by an immunogen.

[0436] As used herein, the term “immunogen” refers to any molecule that is capable of eliciting an immune response. Non-limiting examples of immunogens include immunogenic delivery vehicles such as viral vectors also termed herein “viral particles” (e.g., viral vectors derived from adeno-associated viruses (AAV), adenoviruses, retroviruses [e.g., lentiviruses], or oncolytic viruses [e.g., an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus]) or portions thereof (e.g., capsid proteins), virus-like particles (VLPs), non-viral vectors (e.g., bacteriophages [such as lambda (X) bacteriophage, EMBL bacteriophage; bacterial vectors such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5]; eukaryotic vectors [such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG and pSVL]; transposons [such as Sleeping Beauty transposon and PiggyBac transposon]; bacterial vectors, fungal vectors, and protozoal vectors), liposomes, lipid nanoparticles (LNPs), non-lipid nanoparticles, mammalian cells (e.g., allogeneic cells), and other carriers. Non-limiting examples of immunogens also include polypeptide molecules (e.g., proteins [e.g., therapeutic proteins or antibodies or fragments thereof], peptides), polynucleotide molecules (e.g., mRNAs, interfering nucleic acid molecules [RNAi, siRNA, shRNA], miRNAs, antisense oligonucleotides, ribozymes, aptamers, mixmers, or multimers), antigen-binding molecules fused to a payload, as well as naturally occurring or modified bacteria, fungi, protozoa, parasites, helminths, ectoparasites, or other microorganisms (including bacteria, fungi and other microorganisms found in microbiota). Glycans and lipids are further encompassed by the term immunogen as used herein.

[0437] In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid. In some embodiments, the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle. In some embodiments, the immunogen is an immunogenic delivery vehicle and / or transgene product(s).

[0438] In some embodiments, the immunogenic delivery vehicle is a viral vector. In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, a protozoal vector, or a mammalian cell. In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.

[0439] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent may inhibit an immune response by a cell (e.g., an immune cell such as a B cell or a T cell) or by an immune system of a subject (e.g., a human) which can be elicited by an immunogenic delivery vehicle.

[0440] In some embodiments, an immunogenic delivery vehicle, e.g., a viral particle or vector disclosed herein, may comprise, e.g., one or more of a heterologous and / or recombinant nucleotide sequence(s) of interest (e.g., a nucleotide sequence encoding a gene, or portion thereof, desired to be expressed in a cell targeted by the viral particle (e.g., a transgene), which nucleotide sequence of interest may be, e.g., DNA or RNA). In some embodiments, the nucleotide sequence can encode a polypeptide of interest disclosed herein. In various embodiments, the nucleotide sequence can encode a transgene product comprising one or more therapeutic agents (e.g., therapeutic proteins or polypeptides) described herein.

[0441] In one aspect, the present disclosure provides a method for increasing or maintaining the level of AAV transduction in a target cell and / or tissue, e.g., a target cell and / or tissue within or derived from a subject in need thereof, the method comprising contacting the target cell and / or tissue with, and / or administering to the subject, an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent.

[0442] In another aspect, provided herein is a method for increasing or maintaining the level of AAV transduction in a target cell and / or tissue, e.g., a target cell and / or tissue within or derived from a subject in need thereof (e.g., a subject without pre-existing immunity against AAV), the method comprising contacting the target cell and / or tissue with, and / or administering to the subject, an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof. In some embodiments, the level of AAV transduction in the target cell and / or tissue is increased or maintained by inhibiting or preventing an immune response to the AAV in the subject. In some embodiments, the level of AAV transduction is increased or maintained in the target cell and / or tissue by inhibiting antibody responses to the AAV in the subject.

[0443] In some embodiments, the level of AAV transduction in the target cell and / or tissue is increased or maintained by inhibiting or preventing an immune response to the AAV in a subject. As a non-limiting example, the level of AAV transduction in the target cell and / or tissue may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The level of AAV transduction may be increased in the target cell and / or tissue by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The level AAV transduction may be increased in the target cell and / or tissue by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%. In some embodiments, the level of AAV transduction in the target cell and / or tissue is maintained by inhibiting or preventing an immune response to the AAV in the subject.

[0444] In some embodiments, the level of AAV transduction in the target cell and / or tissue is increased or maintained by inhibiting antibody responses to the AAV in a subject. As a nonlimiting example, the level of AAV transduction in the target cell and / or tissue may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The level of AAV transduction may be increased in the target cell and / or tissue by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The level AAV transduction may be increased in the target cell and / or tissue by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%. In some embodiments, the level of AAV transduction in the target cell and / or tissue is maintained by inhibiting antibody responses to the AAV in the subject.

[0445] In one aspect, the present disclosure provides a method for increasing or maintaining the level of a transgene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent. In some embodiments, the method comprises determining the presence of neutralizing antibodies to the immunogen in the subject. In some embodiments, the transgene is delivered to the subject via an immunogenic delivery vehicle (e.g., AAV).

[0446] In another aspect, provided herein is a method for increasing or maintaining the level of a transgene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof. In some embodiments, the transgene is delivered to the subject via an immunogenic delivery vehicle (e.g., AAV). In some embodiments, the subject does not have a pre-existing immunity against the immunogenic delivery vehicle and / or transgene product(s).

[0447] In some embodiments, the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene (i.e., a transgene product). In some embodiments, the level of transgene expression is increased or maintained by inhibiting antibody responses to the polypeptide or polynucleotide encoded by the transgene.

[0448] In some embodiments, the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to the transgene product(s). In some embodiments, the level of transgene expression is increased or maintained by inhibiting antibody responses to the transgene product(s).

[0449] In some embodiments, the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene. As a nonlimiting example, the level of transgene expression may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The level of transgene expression may be increased by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The level of transgene expression may be increased by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0450] In some embodiments, the immune response to the immunogenic delivery vehicle and / or the immune response to the polypeptide or polynucleotide encoded by the transgene may be inhibited by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The immune response to the immunogenic delivery vehicle and / or the immune response to the polypeptide or polynucleotide encoded by the transgene may be inhibited by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The immune response to the immunogenic delivery vehicle and / or the immune response to the polypeptide or polynucleotide encoded by the transgene may be inhibited by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0451] In another aspect, provided herein is a method for inhibiting or preventing an immune response to an immunogen in a subject in need thereof (e.g., a subject without a pre-existing immunity against the immunogen), the method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof.

[0452] In some embodiments, inhibiting or preventing the immune response comprises suppression of numbers and frequencies of immunogen-specific B cells.

[0453] In some embodiments of the methods for inhibiting or preventing an immune response to an immunogen described herein, the inhibiting of the immune response can comprise suppression of numbers and / or frequencies of plasma cells and / or B cells.

[0454] In some embodiments, the number and / or frequency of plasma cells and / or B cells may be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The number and / or frequency of plasma cells and / or B cells may be reduced by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The number and / or frequency of plasma cells and / or B cells may be reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0455] In some embodiments, the total number and / or frequency of plasma cells and / or B cells may be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The total number and / or frequency of plasma cells and / or B cells may be reduced by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The total number and / or frequency of plasma cells and / or B cells may be reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0456] In some embodiments, inhibiting the immune response comprises suppression of immunogen-specific IgG and / or IgM responses.

[0457] In some embodiments, the responses of IgG and / or IgM may be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The responses of IgG may be reduced by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The responses of IgG may be reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0458] In one aspect, the present disclosure provides a method for increasing effectiveness of re-administration of an immunogen to a subject in need thereof, comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent. As a non-limiting example, the effectiveness of readministration of an immunogen may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7% about 8%, about 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more. The effectiveness of re-administration of an immunogen be increased by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The effectiveness of re-administration of an immunogen may be increased by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%.

[0459] In some embodiments, the immunogen re-administration occurs via the same administration route as its prior administration. In some embodiments, the immunogen readministration occurs via a different administration route than its prior administration. In some embodiments, the plasma cell depleting agent, the B cell depleting agent, and / or the immunoglobulin depleting agent is administered before the administration of the immunogen. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogen to the subject. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogenic delivery vehicle to the subject. In some embodiments, the plasma cell depleting agent, the B cell depleting agent, and / or the immunoglobulin depleting agent is administered simultaneously with the administration of the immunogen. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogen to the subject. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogenic delivery vehicle to the subject. In some embodiments, the plasma cell depleting agent, the B cell depleting agent, and / or the immunoglobulin depleting agent is administered after the administration of the immunogen. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogen to the subject. In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogenic delivery vehicle to the subject. In some embodiments, the immunogen is administered two or more times and the plasma cell depleting agent, the B cell depleting agent, and / or the immunoglobulin depleting agent is administered before and / or between each of the administrations of the immunogen. In some embodiments, the immunogen is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogen. In some embodiments, the immunogenic delivery vehicle is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogenic delivery vehicle. In some embodiments, the plasma cell depleting agent is administered after an immune response has been developed. In some embodiments (e.g., if the patient is immunologically naive), the plasma cell depleting agent is administered simultaneously with the administration of the immunogen (e.g., to prevent any plasma cells from persisting after being formed). In some embodiments, the plasma cell depleting agent is administered after the administration of the immunogen, e.g., 2-4 days afterwards as plasma cell formation may be limited during the initial lag period. In some embodiments, such as when the immunogen is administered two or more times, the plasma cell depleting agent is administered before and / or between each of the administrations of the immunogen. Administration of the plasma cell depleting agent shortly after the administration of the immunogen may prevent plasma cell formation and persistence elicited by administration of the immunogen to immunologically naive patients. In some embodiments (e.g., if the patient is immunologically naive), the B cell depleting agent is administered simultaneously with the administration of the immunogen (e.g., to prevent any B cells from persisting after being formed). In some embodiments, the B cell depleting agent is administered after the administration of the immunogen, e.g., 2-4 days afterwards as B cell formation may be limited during the initial lag period. Administration of the B cell depleting agent shortly after the administration of the immunogen may prevent B cell formation and persistence elicited by administration of the immunogen to immunologically naive patients..In some embodiments (e.g., if the patient already has pre-existing immunity), the plasma cell depleting agent is administered before the administration of the immunogen. In some embodiments (e.g., if the patient already has pre-existing immunity), the plasma cell depleting agent is administered again within a short period of the first administration. In some embodiments (e.g., if the patient already has pre-existing immunity), the plasma cell depleting agent is continuously administered throughout the pre-dose and re-dose periods (e.g., to clear plasma cells and keep plasma cell levels low). In some embodiments (e.g., if the patient already has pre-existing immunity), the plasma cell depleting agent is administered prophylactically. Viral particles

[0460] In one aspect, the present disclosure provides for methods for inhibiting or preventing an immune response to a viral vector in a subject in need thereof, the method comprising administering to the subject an effective amount of a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent. The terms “viral vector” and “viral particle” can be used synonymously and interchangeably herein.

[0461] In some embodiments, the plasma cell depleting agent, the B cell depleting agent, and / or the immunoglobulin depleting agent is capable of inhibiting and / or preventing an immune response which can be elicited by a viral particle, or a portion thereof (e.g., a capsid protein). In some embodiments, the viral particle can comprise a viral vector (e.g., an adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, or an oncolytic virus vector) which can comprise one or more of a heterologous and / or recombinant nucleotide sequence(s) of interest (e.g., a nucleotide sequence encoding a gene, or portion thereof, desired to be expressed in a cell targeted by the viral particle (e.g., a transgene), which nucleotide sequence of interest may be, e.g., DNA or RNA). In some embodiments, the nucleotide sequence can encode a polypeptide of interest disclosed herein. In various embodiments, the nucleotide sequence can encode a transgene product comprising one or more therapeutic agents (e.g., therapeutic proteins or polypeptides) described herein. In some embodiments, the nucleotide sequence encodes a therapeutic protein, a suicide gene, an antibody, or a fragment thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, and / or a shRNA molecule. In some embodiments, the nucleotide sequence may encode a growth factor, a neurotrophic factor, a disease modifying muscle protein, and / or a metabolic protein, e.g., for muscle atrophy conditions or metabolic diseases.

[0462] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent described herein is capable of inhibiting and / or preventing an immune response which can be elicited by a vector, e.g., viral vector, or a portion thereof, e.g., an adenovirus-associated virus (AAV) vector.

[0463] A vector can comprise additional sequences such as, for example, replication origins, promoters, and genes encoding antibiotic resistance. Some vectors may be circular. Alternatively, the vector may be linear. The vector can be in the packaged for delivered via a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0464] Some vectors may be circular. Alternatively, the vector may be linear. The vector can be packaged for delivered via a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0465] The vectors can be, for example, viral vectors such as adeno-associated virus (AAV) vectors. The AAV may be any suitable serotype and may be a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). Other exemplary viruses / viral vectors include retroviruses, lentiviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The viruses can integrate into the host genome or alternatively do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and / or packaging). Viruses can cause transient expression or longer-lasting expression. Viral vector may be genetically modified from their wild type counterparts. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some examples, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some examples, the viral vector may have an enhanced transduction efficiency. In some examples, the immune response induced by the virus in a host may be reduced. In some examples, viral genes (such as integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some examples, the viral vector may be replication defective. In some examples, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some examples, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell or population of host cells along with the vector system described herein. In other examples, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some examples, the vector system described herein may also encode the viral components required for virus amplification and packaging.

[0466] Viral vectors can be derived from naturally occurring virus genomes, which typically are modified to be replication incompetent, e.g. non-replicating. Non-replicating viruses require the provision of proteins in trans for replication. Typically, those proteins are stably or transiently expressed in a viral producer cell line, thereby allowing replication of the virus. The viral vectors are, thus, typically infectious, and non-replicating. Non-limiting examples of viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 8), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g., vectors derived from cytomegaloviruses, like rhesus cytomegalovirus (RhCMV)), arena virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors (canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retrovirus vectors, lentivirus vectors, simian virus 40 (SV40), bovine papilloma viruses, Epstein-Barr viruses, Moloney murine leukemia viruses, Harvey murine sarcoma viruses, murine mammary tumor viruses, Rous sarcoma viruses, poxvirus viral like particles, baculoviral vectors and bacterial spores.

[0467] Adeno-associated viruses (AAVs) are endemic in multiple species including human and non-human primates (NHPs). At least 12 natural serotypes and hundreds of natural variants have been isolated and characterized to date. See, e.g., Li et al. (2020) Nat. Rev. Genet. 21:255-272, herein incorporated by reference in its entirety for all purposes. AAV particles are naturally composed of a non-enveloped icosahedral protein capsid containing a single-stranded DNA (ssDNA) genome. The DNA genome is flanked by two inverted terminal repeats (ITRs) which serve as the viral origins of replication and packaging signals. The rep gene encodes four proteins required for viral replication and packaging whilst the cap gene encodes the three structural capsid subunits which dictate the AAV serotype, and the Assembly Activating Protein (AAP) which promotes virion assembly in some serotypes.

[0468] Recombinant AAV (rAAV) is currently one of the most commonly used viral vectors used in gene therapy to treat human diseases by delivering therapeutic transgenes to target cells in vivo. Indeed, rAAV vectors are composed of icosahedral capsids similar to natural AAVs, but rAAV virions do not encapsidate AAV protein-coding or AAV replicating sequences. These viral vectors are non-replicating. The only viral sequences required in rAAV vectors are the two ITRs, which are needed to guide genome replication and packaging during manufacturing of the rAAV vector. rAAV genomes are devoid of AAV rep and cap genes, rendering them non-replicating in vivo. rAAV vectors are produced by expressing rep and cap genes along with additional viral helper proteins in trans, in combination with the intended transgene cassette flanked by AAV ITRs.

[0469] In therapeutic rAAV genomes, a gene expression cassette is placed between ITR sequences. Typically, rAAV genome cassettes comprise of a promoter to drive expression of a therapeutic transgene, followed by polyadenylation sequence. The ITRs flanking a rAAV expression cassette can be derived from AAV2, the first serotype to be isolated and converted into a recombinant viral vector. Since then, most rAAV production methods rely on AAV2 Repbased packaging systems. See, e.g., Colella etal. (2017) Mol. Then Methods Clin. Dev. 8:87104, herein incorporated by reference in its entirety for all purposes.

[0470] The specific serotype of a recombinant AAV vector influences its in vivo tropism to specific tissues. AAV capsid proteins are responsible for mediating attachment and entry into target cells, followed by endosomal escape and trafficking to the nucleus. Thus, the choice of serotype when developing a rAAV vector will influence what cell types and tissues the vector is most likely to bind to and transduce when injected in vivo. Several serotypes of rAAVs, including rAAV8, are capable of transducing the liver when delivered systemically in mice, NHPs and humans. See, e.g., Li etal. (2020) Nat. Rev. Genet. 21:255-272, herein incorporated by reference in its entirety for all purposes.

[0471] Once in the nucleus, the ssDNA genome is released from the virion and a complementary DNA strand is synthesized to generate a double-stranded DNA (dsDNA) molecule. Double-stranded AAV genomes naturally circularize via their ITRs and become episomes which will persist extrachromosomally in the nucleus. Therefore, for episomal gene therapy programs, rAAV-delivered rAAV episomes provide long-term, promoter-driven gene expression in non-dividing cells. However, this rAAV-delivered episomal DNA is diluted out as cells divide.

[0472] The ssDNA AAV genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats (ITRs) that allow for synthesis of the complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap can be supplied in trans. In addition to Rep and Cap, AAV can require a helper plasmid containing genes from adenovirus. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and the helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to produce infectious AAV particles. Alternatively, the Rep, Cap, and adenovirus helper genes may be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0473] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their tropism), allowing preferential transduction of specific cell types. The term AAV includes, for example, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrhIO, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. An “AAV vector” as used herein refers to an AAV vector comprising a heterologous sequence not of AAV origin (i.e., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding an exogenous polypeptide of interest. The construct may comprise an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrhIO, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV capsid sequence. In general, the heterologous nucleic acid sequence (the transgene) is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). An AAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV). Examples of serotypes for liver tissue include AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74, and AAVhu.37, and particularly AAV8. In a specific example, the AAV vector comprising the nucleic acid construct can be recombinant AAV8 (rAAV8). A rAAV8 vector as described herein is one in which the capsid is from AAV8. For example, an AAV vector using ITRs from AAV2 and a capsid of AAV8 is considered herein to be a rAAV8 vector.

[0474] Tropism can be further refined through pseudotyping, which is the mixing of a capsid and a genome from different viral serotypes. For example AAV2 / 5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. Use of pseudotyped viruses can improve transduction efficiency, as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains a hybrid capsid from eight serotypes and displays high infectivity across a broad range of cell types in vivo. AAV-DJ8 is another example that displays the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of mutational modifications of AAV6 include S663V and T492V. Other pseudotyped / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0475] To accelerate transgene expression, self-complementary AAV (scAAV) variants can be used. Because AAV depends on the cell’s DNA replication machinery to synthesize the complementary strand of the AAV’s single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAV containing complementary sequences that are capable of spontaneously annealing upon infection can be used, eliminating the requirement for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors can also be used.

[0476] To increase packaging capacity, longer transgenes may be split between two AAV transfer plasmids, the first with a 3’ splice donor and the second with a 5’ splice acceptor. Upon co-infection of a cell, these viruses form concatemers, are spliced together, and the full-length transgene can be expressed. Although this allows for longer transgene expression, expression is less efficient. Similar methods for increasing capacity utilize homologous recombination. For example, a transgene can be divided between two transfer plasmids but with substantial sequence overlap such that co-expression induces homologous recombination and expression of the full-length transgene.

[0477] As further examples, adenovirus vectors may be derived from human adenovirus (Ad) but also from adenoviruses that infect other species, such as bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), canine adenovirus (e.g. CAdV2), porcine adenovirus (e.g. PAdV3 or 5), or adenoviruses that infect great apes, such as Chimpanzee (Pan), Gorilla (Gorilla), Orangutan (Pongo), Bonobo (Pan paniscus) and common chimpanzee (Pan troglodytes). Poxvirus (Poxviridae) vectors may be derived from smallpox virus (variola), vaccinia virus, cowpox virus or monkeypox virus. Exemplary vaccinia viruses are the Copenhagen vaccinia virus (W), New York Attenuated Vaccinia Virus (NYVAC), ALVAC, TROVAC and Modified Vaccinia Ankara (MVA).

[0478] In some embodiments, a plasma cell depleting agent, a B cell depleting agent, and / or an immunoglobulin depleting agent of the present disclosure may inhibit an immune response which may be elicited by a transgene product, e.g., a transgene product (e.g., a therapeutic polypeptide or polynucleotide of interest or disclosed herein which is encoded by the transgene ) comprising one or more therapeutic agents. The one or more therapeutic agents may comprise a therapeutic protein (e.g., a therapeutic polypeptide) and / or a therapeutic nucleic acid. Non-limiting examples of therapeutic agents which may be expressed by a transgene using methods of the present disclosure include, e.g., proteins and polypeptides, antisense RNA, or ribozymes, or any combination thereof. In some embodiments, the heterologous and / or recombinant nucleotide sequence becomes integrated into the cell genome. In some embodiments, the heterologous and / or recombinant nucleotide sequence does not become integrated into the cell genome.

[0479] Examples of therapeutic proteins and polypeptides suitable for expression methods of the present disclosure include human hormones such as growth hormone, prolactin, insulin, luteinizing hormone, calcitonin, follicle stimulating hormone, chorionic gonadotropin or thyroid stimulating hormone; a chemokine including, MIP-ip and RANTES la; a colony stimulating factor, e.g., G-CSF, GM- M-CSF and CSF; growth factors such as IGF-1 and IGF-2; a cytokine, such as interleukin (IL)-1, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15, a-interferons, p-interferons, the y-interferons, LFA-1, tumor necrosis factor, CD3, ICAM-1 and LFA-3; LDL receptor, ornithine transcarbamylase, phenylalanine hydroxylase, and al-antitrypsin.

[0480] Additional examples of sequences expressible using the methods described herein include sequences of Protein S and Gas6, thrombin, acidic fibroblast growth factor (FGF-1), basic FGF (FGF-2), keratinocyte growth factor (KGF), TGF, platelet derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and HGF activators, PSA, nerve cell growth factor (NCGF), glial cell derived nerve growth factor (GDNF), vascular endothelial growth factor (VEGF), Arg-vasopressin, thyroid hormones asoxymethane, triodothyronine, LIF, amphiregulin, soluble thrombomodulin, stem cell factor, osteogenic protein 1, the bone morphogenic proteins, MFG, MGSA, heregulins and melanotropin, human growth hormone, leptin, IL-2, erythropoietin, and thrombopoietin (G-CSF).

[0481] In some embodiments, vectors and / or viral particles described herein may comprise, e.g., genes encoding apoptotic factors, genes encoding cytotoxic molecules, genes encoding anti-apoptotic factors, genes encoding immune-stimulatory molecules, a TNF-a gene, a p53 gene, interferon genes, "suicide genes" (i.e., the genes which cause a cell to kill itself through apoptosis; non-limiting examples of suicide genes include, e.g., herpes simplex virus thymidine kinase (HSV-TK), which converts ganciclovir (GCV) into cytotoxic compounds, Escherichia coli cytosine deaminase, which allows the formation of a cytotoxic chemotherapeutic agent from a non-toxic precursor, Varicella-zoster virus thymidine kinase, deoxycytidine kinase, purine nucleoside phosphorylase, nitroreductase, p-galactosidase, hepatic cytochrome P450-2B1, linamarase, horseradish peroxidase, and carboxypeptidase).

[0482] In some embodiments, a protein or polypeptide encoded by the genes inserted into the vectors and viral particles of the present disclosure can provide one or more antigens or antigenically active fragments thereof associated with, e.g., one or more infectious agents such as a bacteria, virus, parasite, or fungus, or a combination thereof, which may be used to immunize a subject. An active fragment described herein may comprise a polypeptide which contains less than a full-length sequence but that retains sufficien...

Claims

1. A method for inhibiting or preventing an immune response to an immunogen in a subject in need thereof, wherein the subject has pre-existing immunity against the immunogen, said method comprising administering to the subject an effective amount of a plasma cell depleting agent.

2. A method for inhibiting or preventing generation of antibodies to an immunogen in a subject in need thereof, wherein the subject has pre-existing immunity against the immunogen, said method comprising administering to the subject an effective amount of a plasma cell depleting agent.

3. A method for increasing effectiveness of re-administration of an immunogen to a subject in need thereof, wherein the subject has pre-existing immunity against the immunogen, said method comprising administering to the subject an effective amount of a plasma cell depleting agent.

4. The method of claim 3, wherein the immunogen re-administration occurs via the same administration route as its prior administration.

5. The method of claim 3, wherein the immunogen re-administration occurs via a different administration route than its prior administration.

6. The method of any one of claims 1-5, comprising determining the presence of neutralizing antibodies to the immunogen in the subject.

7. The method of any one of claims 1-6, wherein the plasma cell depleting agent is administered before the administration of the immunogen.

8. The method of any one of claims 1-6, wherein the plasma cell depleting agent is administered simultaneously with the administration of the immunogen.

9. The method of any one of claims 1-6, wherein the plasma cell depleting agent is administered after the administration of the immunogen.

10. The method of any one of claims 1-9, wherein the immunogen is administered two or more times and the plasma cell depleting agent is administered before and / or between each of the administrations of the immunogen.

11. The method of any one of claims 1-10, wherein the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.

12. The method of claim 11, wherein the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

13. A method for increasing or maintaining the level of a transgene expression in a subject in need thereof, said method comprising administering to the subject an effective amount of a plasma cell depleting agent.

14. The method of claim 13, comprising determining the presence of neutralizing antibodies to the immunogen in the subject.

15. The method of claim 13 or 14, wherein the transgene is delivered to the subject via an immunogenic delivery vehicle.

16. The method of claim 15, wherein the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene.

17. The method of claim 16, wherein the level of transgene expression is increased or maintained by inhibiting antibody responses to the polypeptide or polynucleotide encoded by the transgene.

18. The method of any one of claims 11-12 and 15-17, wherein the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, a protozoal vector, or a mammalian cell.

19. The method of claim 18, wherein the immunogenic delivery vehicle is a viral vector.

20. A method for increasing effectiveness of administration of a subsequently administered viral vector following administration of an originally administered viral vector in a subject in need thereof, said method comprising administering to the subject an effective amount of a plasmacell depleting agent, wherein the subsequently administered viral vector is of the same or similar viral origin as the originally administered viral vector.

21. The method of claim 20, comprising determining the presence of neutralizing antibodies to the immunogen in the subject.

22. The method of claim 20 or 21, wherein the subsequently administered viral vector is administered via the same administration route as the originally administered viral vector.

23. The method of claim 20 or 21, wherein the subsequently administered viral vector is administered via a different administration route from the originally administered viral vector.

24. The method of any one of claims 20-23, wherein the plasma cell depleting agent is administered before the administration of the subsequently administered viral vector(s).

25. The method of any one of claims 20-23, wherein the plasma cell depleting agent is administered simultaneously with the administration of the subsequently administered viral vector(s).

26. The method of any one of claims 20-23, wherein the subsequently administered viral vectors are administered two or more times and the plasma cell depleting agent is administered before and / or between each of the administrations of the subsequently administered viral vectors.

27. The method of any one of claims 20-26, wherein the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, a retrovirus, or an oncolytic virus.

28. The method of claim 27, wherein the viral vector is AAV.

29. The method of any one of claims 1-28, wherein the plasma cell depleting agent is capable of depleting long-lived plasma cells (LLPC).

30. The method of any one of claims 1-29, wherein the plasma cell depleting agent is a B cell maturation antigen (BCMA) targeting agent.

31. The method of claim 30, wherein the BCMA targeting agent is a chimeric antigen receptor (CAR) against BCMA or an anti-BCMA antibody or a functional fragment thereof.

32. The method of claim 31, wherein the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent.

33. The method of claim 31 or 32, wherein the anti-BCMA antibody is a multispecific antibody or a functional fragment thereof.

34. The method of claim 33, wherein the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3.

35. The method of claim 34, wherein the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof.

36. The method of claim 35, wherein the anti-BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

37. The method of claim 35, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18.

38. The method of claim 37, wherein the first antigen-binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

39. The method of claim 37 or 38, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 andLCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18.

40. The method of claim 39, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

41. The method of any one of claims 37-40, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively.

42. The method of claim 41, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 22, and 24, respectively.

43. The method of any one of claims 35-42, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

44. The method of claim 43, wherein the human IgG heavy chain constant region is isotype lgG4 or lgG1.

45. The method of claim 43 or 44, wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

46. The method of claim 43 or 44, wherein the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

47. The method of any one of claims 1-46, further comprising administering to the subject an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent.

48. The method of claim 47, wherein the B cell depleting agent is administered before, at the same time as, or after the plasma cell depleting agent.

49. The method of claim 47 or 48, wherein the immunoglobulin depleting agent is administered after the plasma cell depleting agent.

50. The method of any one of claims 47-49, wherein the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

51. The method of any one of claims 47-50, wherein the B cell depleting agent is an agent that binds to a B cell surface molecule.

52. The method of claim 51, wherein the B cell depleting agent is selected from anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD79 antibodies, multispecific antibodies combining two or more of any of said antibody specificities, multispecific antibodies combining any of said antibody specificities with anti-CD3 antibodies, functional fragments of any of said antibodies, and any combinations thereof.

53. The method of claim 52, wherein the B cell depleting agent comprises (i) an anti-CD20 antibody or a functional fragment thereof and (ii) an anti-CD19 antibody or a functional fragment thereof.

54. The method of claim 52, wherein the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof.

55. The method of claim 54, wherein the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof.

56. The method of claim 55, wherein the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3.

57. The method of claim 56, wherein the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

58. The method of claim 57, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

59. The method of claim 58, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

60. The method of claim 58 or 59, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

61. The method of claim 60, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO:50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

62. The method of any one of claims 58-61, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

63. The method of any one of claims 57-62, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.

64. The method of claim 63, wherein the human IgG heavy chain constant region is isotype lgG4 or lgG1.

65. The method of claim 63 or 64, wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).

66. The method of claim 63 or 64, wherein the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

67. The method of any one of claims 47-50, wherein the B cell depleting agent is an agent targeting a B cell survival factor.

68. The method of any one of claims 47-50, wherein the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof.

69. The method of any one of claims 47-68, wherein the immunoglobulin depleting agent is capable of accelerating IgG clearance.

70. The method of any one of claims 47-69, wherein the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker.

71. The method of claim 70, wherein the FcRn blocker is selected from Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), Nipocalimab (M281), Orilanolimab (SYNT001), IMVT-1402, and any combinations thereof.

72. The method of any one of claims 1-71, wherein the method further comprises plasmapheresis, therapeutic plasma exchange, or immunoadsorption.

73. A pharmaceutical composition comprising (i) a plasma cell depleting agent, (ii) a B cell depleting agent and / or an immunoglobulin depleting agent, and (iii) a pharmaceutically acceptable carrier and / or excipient.

74. A pharmaceutical composition comprising (i) an immunogen, (ii) a plasma cell depleting agent, (iii) optionally, a B cell depleting agent and / or an immunoglobulin depleting agent, and (iv) a pharmaceutically acceptable carrier and / or excipient.

75. A kit comprising (i) a plasma cell depleting agent, (ii) a B cell depleting agent and / or an immunoglobulin depleting agent, and (iii) optionally, instructions for use.

76. A kit comprising (i) an immunogen, (ii) a plasma cell depleting agent, (iii) optionally a B cell depleting agent and / or an immunoglobulin depleting agent, and (iv) optionally, instructions for use.

77. A method for inhibiting or preventing an immune response to an immunogen in a subject in need thereof, said method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof.

78. The method of claim 77, wherein inhibiting the immune response comprises suppression of numbers and frequencies of immunogen-specific B cells.

79. The method of claim 77 or 78, wherein inhibiting the immune response comprises suppression of immunogen-specific IgG and / or IgM responses.

80. A method for inhibiting or preventing generation of neutralizing antibodies to an immunogen in a subject in need thereof, said method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof.

81. A method for increasing effectiveness of re-administration of an immunogen to a subject in need thereof, said method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof.

82. The method of claim 81, wherein the immunogen re-administration occurs via the same administration route as its prior administration.

83. The method of claim 81, wherein the immunogen re-administration occurs via a different administration route than its prior administration.

84. The method of any one of claims 77-83, wherein the subject does not have a pre-existing immunity against the immunogen.

85. The method of any one of claims 77-84, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogen to the subject.

86. The method of any one of claims 77-84, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogen to the subject.

87. The method of any one of claims 77-84, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogen to the subject.

88. The method of any one of claims 77-84, wherein the immunogen is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogen.

89. The method of any one of claims 77-88, wherein the immunogen is an immunogenic delivery vehicle, a polypeptide, ,a polynucleotide, a glycan, or a lipid.

90. The method of claim 89, wherein the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.

91. A method for increasing or maintaining the level of a transgene expression in a subject in need thereof, said method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof.

92. The method of claim 91, wherein the transgene is delivered to the subject via an immunogenic delivery vehicle.

93. The method of claim 92, wherein the level of transgene expression is increased or maintained by inhibiting an immune response to the immunogenic delivery vehicle and / or by inhibiting an immune response to a polypeptide or polynucleotide encoded by the transgene.

94. The method of claim 92, wherein the level of transgene expression is increased or maintained by inhibiting antibody responses to a polypeptide or polynucleotide encoded by the transgene.

95. The method of any one of claims 92-94, wherein the subject does not have a pre-existing immunity against the immunogenic delivery vehicle and / or a polypeptide or polynucleotide encoded by the transgene.

96. The method of any one of claims 92-95, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the immunogenic delivery vehicle to the subject.

97. The method of any one of claims 92-95, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the immunogenic delivery vehicle to the subject.

98. The method of any one of claims 92-95, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the immunogenic delivery vehicle to the subject.

99. The method of any one of claims 92-95, wherein the immunogenic delivery vehicle is administered to the subject two or more times and the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the immunogenic delivery vehicle.100.The method of any one of claims 92-99, wherein the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.101 .The method of claim 100, wherein the immunogenic delivery vehicle is a viral vector.102.A method for increasing effectiveness of a subsequently administered viral vector following an originally administered viral vector in a subject in need thereof, said method comprising administering to the subject an effective amount of an anti-CD20xCD3 bispecific antibody or a functional fragment thereof, wherein the subsequently administered viral vector is of the same or similar viral origin as the originally administered viral vector.103.The method of claim 102, wherein the subsequently administered viral vector is administered via the same administration route as the originally administered viral vector.104.The method of claim 102, wherein the subsequently administered viral vector is administered via a different administration route from the originally administered viral vector.105.The method of any one of claim 102-104, wherein the subject does not have a pre-existing immunity against the viral vectors.106.The method of any one of claims 102-105, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before the administration of the originally administered viral vector to the subject.107.The method of any one of claims 102-105, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the administration of the originally administered viral vector and / or subsequently administered viral vector to the subject.108.The method of any one of claims 102-105, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the originally administered viral vector but before administering the subsequently administered viral vector to the subject.109.The method of any one of claims 102-105, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered after the administration of the subsequently administered viral vector to the subject.110.The method of any one of claims 102-105, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered before and / or between each of the administrations of the viral vectors to the subject.111.The method of any one of claims 102-110, wherein the viral vectors are derived from an adeno-associated virus (AAV), an adenovirus, ora retrovirus.112.The method of claim 111, wherein the viral vectors are derived from AAV.113.The method of claim 112, wherein the subsequently administered AAV vector has a capsid derived from the same AAV serotype as the originally administered AAV vector.114.The method of claim 111, wherein the retrovirus is a lentivirus.115.The method of any one of claims 102-110, wherein the viral vectors are derived from an oncolytic virus.116.The method of claim 115, wherein the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.117.The method of any one of claims 77-116, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.118.The method of claim 117, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO:50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.119.The method of claim 117 or 118, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.120.The method of claim 119, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.121.The method of any one of claims 117-120, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.122.The method of any one of claims 117-121, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.123.The method of claim 122, wherein the human IgG heavy chain constant region is isotype lgG4 or lgG1.124.The method of claim 122 or 123, wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).125.The method of claim 122 or 123, wherein the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).126.A composition comprising an immunogen and an anti-CD20xCD3 bispecific antibody or a functional fragment thereof and optionally further comprising a pharmaceutically acceptable carrier and / or excipient.127.The composition of claim 126, wherein the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.128.The composition of claim 127, wherein the immunogen is an immunogenic delivery vehicle ora polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.129.The composition of claim 127, wherein the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.130.The composition of claim 129, wherein the immunogenic delivery vehicle is a viral vector.131.The composition of claim 130, wherein the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, or a retrovirus.132.The composition of claim 131, wherein the viral vector is derived from AAV.133.The composition of claim 131, wherein the retrovirus is a lentivirus.134.The composition of claim 130, wherein the viral vector is derived from an oncolytic virus.135.The composition of claim 134, wherein the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.136.The composition of any one of claims 126-135, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequenceof SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.137.The composition of claim 136, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.138.The composition of claim 136 or 137, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.139.The composition of claim 138, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.140.The composition of any one of claims 136-139, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.141.The composition of any one of claims 136-140, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.142.The composition of claim 141, wherein the human IgG heavy chain constant region is isotype lgG4 or lgG1.143.The composition of claim 141 or 142, wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).144.The composition of claim 141 or 142, wherein the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).145.A kit comprising (i) an immunogen, (ii) an anti-CD20xCD3 bispecific antibody or a functional fragment thereof, and (iii) optionally, instructions for use.146.The kit of claim 145, wherein the immunogen is an immunogenic delivery vehicle, a polypeptide, a polynucleotide, a glycan, or a lipid.147.The kit of claim 146, wherein the immunogen is an immunogenic delivery vehicle or a polypeptide or polynucleotide encoded by a transgene contained within the immunogenic delivery vehicle.148.The kit of claim 146, wherein the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector.149.The kit of claim 148, wherein the immunogenic delivery vehicle is a viral vector.150.The kit of claim 149, wherein the viral vector is derived from an adeno-associated virus (AAV), an adenovirus, or a retrovirus.151.The kit of claim 150, wherein the viral vector is derived from AAV.152.The kit of claim 150, wherein the retrovirus is a lentivirus.153.The kit of claim 149, wherein the viral vector is derived from an oncolytic virus.154.The kit of claim 153, wherein the oncolytic virus is an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus.155.The kit of any one of claims 145-154, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.156.The kit of claim 155, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.157.The kit of claim 155 or 156, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.158.The kit of claim 157, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.159.The kit of any one of claims 155-158, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises:a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1,LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; andb) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.160.The kit of any one of claims 155-159, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region.161.The kit of claim 160, wherein the human IgG heavy chain constant region is isotype lgG4 or lgG1.162.The kit of claim 160 or 161, wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn).163.The kit of claim 160 or 161, wherein the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcyR).

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