RNA Composition Targeting HIV

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for the delivery of anti-HIV antibody agents, and related technologies (e.g., its components, and / or methods related thereto). In particular among others, the present disclosure provides polynucleotides encoding immunoglobulin chains of anti-HIV antibody agents. The present disclosure also provides technologies that address certain limitations of recombinant antibody technology, including, for example, the short serum half-life of recombinant antibodies, by utilizing RNA technology as a mode for directly expressing antibody agents within the cells of a patient.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 342,057, filed on May 13, 2022, the entire contents of which are incorporated herein by reference.

Background Art

[0002] The human immunodeficiency virus (HIV) is an infectious virus associated with acquired immunodeficiency syndrome (AIDS). According to the World Health Organization, more than 37 million people worldwide are currently infected with HIV. In 2020, approximately 680,000 people died from HIV - related causes, and approximately 1.5 million people were newly infected with HIV. Currently, there is no cure for HIV.

Summary of the Invention

Means for Solving the Problems

[0003] The present disclosure recognizes that HIV mutates rapidly. The property of frequent mutation of HIV virions enables them to escape from host immunity and / or treatment pressure. To counter viral escape, combination therapies targeting HIV, including anti-HIV antibody agents, have been investigated. However, the development of such combination therapies has been hindered by multiple challenges. First, the development of individual HIV therapies is time-consuming and costly. For example, in the development of anti-HIV antibodies, production requires much, including purification and formulation methods related to protein therapeutics, and the cost is a problem. Second, combination therapies present regulatory challenges. Not only ensuring that combination therapies are safe and effective, but also strict regulations regarding the manufacture of individual therapies, the compounding of multiple therapies, and quality control during storage and administration make the use of combination therapies complex. Third, the administration of antibodies to a subject can be painful and time-consuming. Generally, antibodies are administered intravenously over a longer period. The administration of multiple antibodies can increase the complexity of antibody administration, which can result in promoting patient discomfort and requiring additional time. Finally, recombinant antibodies may have a short serum half-life.

[0004] The present disclosure provides an ocular delivery that addresses these issues and enables the safe, reliable, and potent delivery of a single anti-HIV antibody agent to a subject, and also enables the delivery of multiple anti-HIV therapeutic agents, including multiple anti-HIV antibody agents, to a subject. For example, the present disclosure describes an antibody agent or a portion thereof (e.g., an immunoglobulin chain) that is delivered to a subject via a polynucleotide. An antibody agent that is delivered to a subject as one or more polynucleotides encoding the antibody agent is referred to herein as a "RiboMab". After delivering one or more polynucleotides encoding the antibody agent to a subject, the antibody agent, i.e., the "RiboMab", is expressed by the subject's body. Further, the term "RibobNAb" refers to a RiboMab that includes all or a portion of a broadly neutralizing antibody (bNAb), e.g., a broadly neutralizing antibody targeting HIV. Utilizing polynucleotides as therapeutic agents involves a simpler and less expensive manufacturing process (as opposed to administering the antibody agent itself). The simpler production of polynucleotides encoding an antibody agent(s) (e.g., an anti-HIV antibody agent(s)) enables the streamlining of manufacturing (e.g., by avoiding the need for intensive glycan production and characterization), and as a result, reduces regulatory and production challenges associated with developing and using the antibody agent itself. In addition, polynucleotides tend to require far less amount to be administered to a subject while still being effective in producing effects similar to recombinant proteins. This is because, for example, polynucleotides encoding an anti-HIV antibody agent(s) can be administered to a subject, and the subject's body produces the anti-HIV antibody agent(s) itself. By reducing the amount used, a more comfortable experience for the patient can be provided, and patient compliance with the treatment plan can be enhanced. The present disclosure also provides a technique that addresses certain limitations of recombinant antibody technology, including, for example, the short serum half-life of recombinant antibodies, by utilizing RNA technology as a mode for directly expressing an antibody agent within a patient's cells.

[0005] The RiboMab technology also enables the simultaneous administration of two or more antibody agents. For example, antibodies produced by humans typically comprise four polypeptide chains, two "heavy" chains and two "light" chains. Each polypeptide chain (regardless of whether it is a heavy or light chain) contains (1) a "variable" domain that has a sequence that is different for each antibody and a structure that determines the antigen to which the antibody binds, and (2) one or more "constant" domains that have a sequence and structure that generally remain invariant across a given class of antibodies and thus have little effect on antigen binding. In humans, "B cells", a type of specialized white blood cell, produce antibodies. The heavy and light chains associate to form an antibody through two main pairings: (1) the crystallizable fragment (Fc) domains of the two heavy chains pair with each other, and (2) each of the two light chains pairs with a heavy chain via a disulfide bond. During normal antibody production in humans, a single antibody is produced by a single B cell. In that situation, since there is only one type of heavy chain and one type of light chain within each B cell, the correct pairing of the heavy and light chains is ensured. In contrast, administering a nucleic acid composition encoding more than one type of antibody agent requires correctly associating the immunoglobulin chains (e.g., heavy and light chains) so that unwanted by-products (e.g., antibody agents in unintended paired states) are not formed.

[0006] In particular, the present disclosure provides polyribonucleotides encoding the immunoglobulin chains of antibody agents.

[0007] In one aspect, the immunoglobulin chain comprises a variable heavy (VH) domain. In some embodiments, the VH domain comprises a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence according to SEQ ID NO: 6, an HCDR2 having the amino acid sequence according to SEQ ID NO: 9, and an HCDR3 having the amino acid sequence according to SEQ ID NO: 12.

[0008] In some embodiments, the VH domain comprises or consists of the amino acid sequence according to SEQ ID NO: 24. In some embodiments, the VH domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 24.

[0009] In some embodiments, the polynucleotide comprises a VH domain coding sequence. In some embodiments, the VH domain coding sequence comprises (a) an HCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 7, (b) an HCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 10, and (c) an HCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 13.

[0010] In some embodiments, the VH domain coding sequence comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 25. In some embodiments, the VH domain coding sequence comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 25. In some embodiments, the VH domain coding sequence comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 27. In some embodiments, the VH domain coding sequence comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 27.

[0011] In some embodiments, the immunoglobulin chain comprising the VH domain described herein comprises one or more constant domains. In some embodiments, the VH domain is operably linked to one or more constant domains.

[0012] In some embodiments, the immunoglobulin chain comprising the VH domain described herein comprises one or more constant domains. In some embodiments, the VH domain is operably linked to one or more constant domains.

[0013] In some embodiments, the one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain.

[0014] In some embodiments, the one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH3 domain.

[0015] In some embodiments, the one or more constant domains comprise a hinge domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the hinge domain.

[0016] In some embodiments, the one or more constant domains comprise a CH1 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH1 domain.

[0017] In some embodiments, the one or more constant domains comprise a CL domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CL domain.

[0018] In some embodiments, the immunoglobulin chain comprising the VH domain described herein comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain.

[0019] In some embodiments, the immunoglobulin chain comprising the VH domain described herein comprises a CL domain, a hinge domain, a CH2 domain, and a CH3 domain.

[0020] The present disclosure further provides a polynucleotide encoding an immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a variable light (VL) domain.

[0021] In some embodiments, the VL domain comprises (a) light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence according to SEQ ID NO: 15, (b) LCDR2 comprising the amino acid sequence (GTS) according to SEQ ID NO: 18, and (c) LCDR3 comprising the amino acid sequence according to SEQ ID NO: 21.

[0022] In some embodiments, the polynucleotide comprises a VL domain coding sequence. In some embodiments, the VL domain coding sequence comprises (a) an LCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 16, an LCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence (GGCACCAGC) according to SEQ ID NO: 19, and an LCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 22.

[0023] In some embodiments, the VL domain comprises or consists of the amino acid sequence according to SEQ ID NO: 29. In some embodiments, the VL domain coding sequence comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 30.

[0024] In some embodiments, the immunoglobulin chain comprising the VL domain further comprises a constant domain. In some embodiments, the VL domain is operably linked to the constant domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the constant domain.

[0025] In some embodiments, an immunoglobulin chain comprising a VL domain further comprises a CL domain. In some embodiments, a polynucleotide comprises a ribonucleic acid sequence encoding a CL domain. In some embodiments, the CL domain is a kappa constant domain. In some embodiments, a polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain.

[0026] In some embodiments, an immunoglobulin chain comprising a VL domain further comprises a CH1 domain. In some embodiments, a polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain.

[0027] In particular, the present disclosure provides a polynucleotide encoding an immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain comprises HCDR1 having the amino acid sequence according to SEQ ID NO: 6, HCDR2 having the amino acid sequence according to SEQ ID NO: 9, and HCDR3 having the amino acid sequence according to SEQ ID NO: 12. In some embodiments, the VL domain comprises LCDR1 having the amino acid sequence according to SEQ ID NO: 15, LCDR2 having the amino acid sequence (GTS) according to SEQ ID NO: 18, and LCDR3 having the amino acid sequence according to SEQ ID NO: 21.

[0028] In some embodiments, the polynucleotide comprises a VH domain coding sequence and a VL domain coding sequence. In some embodiments, the VH domain coding sequence comprises an HCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 7, an HCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 10, and an HCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 13. In some embodiments, the VL domain coding sequence comprises an LCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 16, an LCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence (GGCACCAGC) according to SEQ ID NO: 19, and an LCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO: 22.

[0029] In some embodiments, the immunoglobulin chain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a VH domain, a linker, and a VL domain.

[0030] In some embodiments, the scFv comprises a VH domain, a linker, and a VL domain in that order. In some embodiments, the scFv comprises, in order, a VH domain comprising or consisting of the amino acid sequence according to SEQ ID NO: 24, a linker, and a VL domain comprising or consisting of the amino acid sequence according to SEQ ID NO: 29.

[0031] In some embodiments, the scFv comprises a VL domain, a linker, and a VH domain in that order. In some embodiments, the scFv comprises, in order, a VL domain comprising or consisting of the amino acid sequence according to SEQ ID NO: 29, a linker, and a VH domain comprising or consisting of the amino acid sequence according to SEQ ID NO: 24.

[0032] In some embodiments, the linker comprises the amino acid sequence according to SEQ ID NO: 32. In some embodiments, the polynucleotide encodes a linker and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 33.

[0033] In some embodiments, the linker comprises the amino acid sequence according to SEQ ID NO: 35. In some embodiments, the polynucleotide encodes a linker and comprises or consists of a ribonucleic acid sequence comprising the sequence according to SEQ ID NO: 36.

[0034] In some embodiments, the immunoglobulin chain comprising the VH domain and VL domain described herein comprises one or more constant domains. In some embodiments, the VH domain and VL domain are operably linked to one or more constant domains.

[0035] In some embodiments, the immunoglobulin chain comprises one or more constant domains, and the hinge domain is between the scFv and the one or more constant domains.

[0036] In some embodiments, the one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain.

[0037] In some embodiments, the one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH3 domain.

[0038] In some embodiments, the one or more constant domains comprise a hinge domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the hinge domain.

[0039] In some embodiments, the one or more constant domains comprise a CH1 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH1 domain.

[0040] According to the present disclosure, any CH2 domain of the above embodiments may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 53.

[0041] In some embodiments, the ribonucleic acid sequence encoding the CH2 domain may include a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 54. In some embodiments, the ribonucleic acid sequence encoding the CH2 domain comprises or consists of the sequence according to SEQ ID NO: 54.

[0042] In some embodiments, the CH2 domain includes one or more substitution mutations. In some embodiments, one or more substitution mutations in the CH2 domain comprise or consist of G236A, A330L, I332E or a combination thereof, and the substitution mutation positions are based on the EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain comprise or consist of G236A, and the substitution mutation position is based on the EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain comprise or consist of I332E, and the substitution mutation position is based on the EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain comprise or consist of G236A and I332E, and the substitution mutation position is based on the EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain comprise or consist of G236A, A330L and I332E, and the substitution mutation position is based on the EU numbering.

[0043] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 56. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 56.

[0044] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain, having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 57. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain and comprising or consisting of the sequence according to SEQ ID NO: 57.

[0045] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 59. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 59.

[0046] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 60. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain and comprising or consisting of the sequence according to SEQ ID NO: 60.

[0047] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 62. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 62.

[0048] In some embodiments, the polynucleotide encodes a CH2 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide encodes a CH2 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence of SEQ ID NO: 63.

[0049] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 65.

[0050] In some embodiments, the polynucleotide encodes a CH2 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 66. In some embodiments, the polynucleotide encodes a CH2 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence of SEQ ID NO: 66.

[0051] According to the present disclosure, any CH3 domain of the above embodiments may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 68. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 68.

[0052] In some embodiments, the polynucleotide encodes a CH3 domain and includes a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 69. In some embodiments, the polynucleotide encodes a CH3 domain and includes a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 69.

[0053] In some embodiments, the CH3 domain includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 71. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 71.

[0054] In some embodiments, the polynucleotide encodes a CH3 domain and includes a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 72. In some embodiments, the polynucleotide encodes a CH3 domain and includes a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 72.

[0055] In some embodiments, the CH3 domain comprises one or more substitution mutations. In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of M428L, N434S, or a combination thereof, and the substitution mutation positions are based on EU numbering.

[0056] In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 74. In some embodiments, the polynucleotide encodes the CH3 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 75.

[0057] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence according to SEQ ID NO: 77. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 77.

[0058] In some embodiments, the polynucleotide encodes the CH3 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the ribonucleic acid sequence according to SEQ ID NO: 78. In some embodiments, the polynucleotide encodes the CH3 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 78.

[0059] In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of Y349C, T366S, L368A, Y407V, or combinations thereof, and the substitution mutation positions are based on EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of Y349C, T366S, L368A, Y407V, M428L, N434S, or combinations thereof, and the substitution mutation positions are based on EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of Y349C, T366S, L368A, Y407V, M428L, and N434S, and the substitution mutation positions are based on EU numbering.

[0060] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence according to SEQ ID NO: 80. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 80.

[0061] In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the ribonucleic acid sequence according to SEQ ID NO: 81. In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 81.

[0062] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence according to SEQ ID NO: 83. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 83.

[0063] In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 84. In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 84.

[0064] In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of S354C, T366W or a combination thereof, and the substitution mutation positions are based on EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of S354C, T366W, M428L, N434S or a combination thereof, and the substitution mutation positions are based on EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise or consist of S354C, T366W, M428L and N434S, and the substitution mutation positions are based on EU numbering.

[0065] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 86. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 86.

[0066] In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 87. In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 87.

[0067] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 89. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 89.

[0068] In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 90. In some embodiments, the polynucleotide encodes a CH3 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 90.

[0069] According to the present disclosure, any hinge domain of the above embodiments may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 104. In some embodiments, the hinge domain comprises or consists of the amino acid sequence according to SEQ ID NO: 104.

[0070] In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 105. In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 105.

[0071] In some embodiments, the hinge domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the hinge domain comprises or consists of the amino acid sequence of SEQ ID NO: 110.

[0072] In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the ribonucleic acid sequence of SEQ ID NO: 111. In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence of SEQ ID NO: 111.

[0073] In some embodiments, the hinge domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the hinge domain comprises or consists of the amino acid sequence of SEQ ID NO: 107.

[0074] In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the ribonucleic acid sequence of SEQ ID NO: 108. In some embodiments, the polynucleotide encodes a hinge domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence of SEQ ID NO: 108.

[0075] According to the present disclosure, any CH1 domain of the above embodiments may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 38. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 38.

[0076] In some embodiments, the polynucleotide encodes a CH1 domain and includes a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 39. In some embodiments, the polynucleotide encodes a CH1 domain and includes a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 39.

[0077] In some embodiments, the CH1 domain includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 41. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 41.

[0078] In some embodiments, the polynucleotide encodes a CH1 domain and includes a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 42. In some embodiments, the polynucleotide encodes a CH1 domain and includes a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 42.

[0079] In some embodiments, the CH1 domain comprises one or more substitution mutations. In some embodiments, one or more substitution mutations in the CH1 domain comprise or consist of K147E, K213D, or a combination thereof, and the substitution mutation positions are based on EU numbering.

[0080] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence according to SEQ ID NO: 50. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 50.

[0081] In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the ribonucleic acid sequence according to SEQ ID NO: 51. In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence according to SEQ ID NO: 51.

[0082] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO: 44.

[0083] In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 45. In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 45.

[0084] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 47.

[0085] In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a CH1 domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 48.

[0086] According to the present disclosure, any CL domain of the above embodiments may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 92. In some embodiments, any CL domain of the above embodiments may comprise or consist of the amino acid sequence of SEQ ID NO: 92.

[0087] In some embodiments, the polynucleotide encodes a CL domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 93. In some embodiments, the polynucleotide encodes a CL domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 93.

[0088] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain, and the CL domain comprises one or more substitution mutations. In some embodiments, one or more substitution mutations in the CL domain comprise or consist of Q124E, and the substitution mutation position is based on the EU numbering. In some embodiments, one or more substitution mutations in the CL domain comprise or consist of R108A, T109S or a combination thereof, and the substitution mutation position is based on the EU numbering. In some embodiments, one or more substitution mutations in the CL domain comprise or consist of R108A, T109S, Q124E or a combination thereof, and the substitution mutation position is based on the EU numbering.

[0089] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 95. In some embodiments, the CL domain comprises or consists of the amino acid sequence of SEQ ID NO: 95.

[0090] In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 96. In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 96.

[0091] In some embodiments, the CL domain comprises one or more substitution mutations. In some embodiments, one or more substitution mutations in the CL domain comprise or consist of E123K, Q124R or a combination thereof, and the substitution mutation positions are based on EU numbering.

[0092] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 98. In some embodiments, the CL domain comprises or consists of the amino acid sequence according to SEQ ID NO: 98.

[0093] In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO: 99. In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence comprising or consisting of the sequence according to SEQ ID NO: 99.

[0094] In some embodiments, one or more substitution mutations in the CL domain comprise or consist of E123R, Q124K or a combination thereof, and the substitution mutation positions are based on EU numbering.

[0095] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 101. In some embodiments, the CL domain comprises or consists of the amino acid sequence of SEQ ID NO: 101.

[0096] In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the ribonucleic acid sequence of SEQ ID NO: 102. In some embodiments, the polynucleotide encodes a light chain constant domain and comprises a ribonucleic acid sequence that comprises or consists of the sequence of SEQ ID NO: 102.

[0097] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains, (i) the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain, the CH2 domain comprises one or more substitution mutations, and the one or more substitution mutations comprise or consist of G236A, A330L, I332E or combinations thereof, (ii) the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain, the CH3 domain comprises one or more substitution mutations, and the one or more substitution mutations comprise or consist of Y349C, S354C, T366S, T366W, L368A, Y407V, M428L, N434S or combinations thereof, (iii) the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain, the CH1 domain comprises one or more substitution mutations, and the one or more substitution mutations comprise or consist of K147E, K213D or combinations thereof, (iv) The polynucleotide comprises a ribonucleic acid sequence encoding a CL domain, the CL domain comprises one or more substitution mutations, and the one or more substitution mutations may comprise or consist of R108A, T109S, E123K, E123R, Q124E, Q124K, Q124R or combinations thereof, or (v) combinations thereof, The substitution mutation positions are based on EU numbering.

[0098] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 614. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 614.

[0099] In some embodiments, the polynucleotides provided herein comprise a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the ribonucleic acid sequence of SEQ ID NO: 613. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 613.

[0100] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 617. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 617.

[0101] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 616. In some embodiments, the polynucleotides provided herein include or consist of a ribonucleic acid sequence according to SEQ ID NO: 616.

[0102] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 623. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence according to SEQ ID NO: 623.

[0103] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 622. In some embodiments, the polynucleotides provided herein include or consist of a ribonucleic acid sequence according to SEQ ID NO: 622.

[0104] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 626. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence according to SEQ ID NO: 626.

[0105] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 625. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence according to SEQ ID NO: 625.

[0106] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 635. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence according to SEQ ID NO: 635.

[0107] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 634. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence according to SEQ ID NO: 634.

[0108] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 641. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence according to SEQ ID NO: 641.

[0109] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 640. In some embodiments, the polynucleotide provided herein comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 640.

[0110] In some embodiments, the polynucleotide provided herein encodes an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 644. In some embodiments, the polynucleotide provided herein encodes an immunoglobulin chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 644.

[0111] In some embodiments, the polynucleotide provided herein comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 643. In some embodiments, the polynucleotide provided herein comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 643.

[0112] In some embodiments, the polynucleotide provided herein encodes an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 647. In some embodiments, the polynucleotide provided herein encodes an immunoglobulin chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 647.

[0113] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 646. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 646.

[0114] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 650. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 650.

[0115] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 649. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 649.

[0116] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 653. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 653.

[0117] In some embodiments, the polynucleotides provided herein comprise a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 652. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 652.

[0118] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 629. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 629.

[0119] In some embodiments, the polynucleotides provided herein comprise a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 628. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence of SEQ ID NO: 628.

[0120] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 620. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 620.

[0121] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 619. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 619.

[0122] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 638. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 638.

[0123] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 637. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 637.

[0124] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 668. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 668.

[0125] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 667. In some embodiments, the polynucleotides provided herein comprise or consist of a ribonucleic acid sequence of SEQ ID NO: 667.

[0126] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 632. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the amino acid sequence of SEQ ID NO: 632.

[0127] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 631. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence of SEQ ID NO: 631.

[0128] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 656. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the sequence of SEQ ID NO: 656.

[0129] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 655. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence of SEQ ID NO: 655.

[0130] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 659. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the sequence of SEQ ID NO: 659.

[0131] In some embodiments, the polynucleotides provided herein comprise ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 658. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence of SEQ ID NO: 658.

[0132] In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 662. In some embodiments, the polynucleotides provided herein encode an immunoglobulin chain comprising or consisting of the sequence of SEQ ID NO: 662.

[0133] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 661. In some embodiments, the polynucleotides provided herein include or consist of the ribonucleic acid sequence of SEQ ID NO: 661.

[0134] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising amino acid sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 665. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the sequence of SEQ ID NO: 665.

[0135] In some embodiments, the polynucleotides provided herein include ribonucleic acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 664. In some embodiments, the polynucleotides provided herein include or consist of the ribonucleic acid sequence of SEQ ID NO: 664.

[0136] In some embodiments, the polynucleotide includes a ribonucleic acid sequence encoding a secretion signal.

[0137] In some embodiments, the secretion signal includes the ribonucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0138] In some embodiments, the polynucleotide includes one or more non-coding sequence elements.

[0139] In some embodiments, one or more non-coding sequence elements improve the stability and / or translation efficiency of the RNA.

[0140] In some embodiments, one or more non-coding sequence elements include a 3' untranslated region (UTR), 5' UTR, 5' cap, polyadenine (polyA) tail, or a combination thereof.

[0141] In some embodiments, the polyA tail is or includes a modified polyA sequence, preferably an interrupted polyA tail.

[0142] In some embodiments, the polyA tail comprises or consists of a sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 474.

[0143] In some embodiments, the 3' UTR comprises or consists of a nucleic acid sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 473.

[0144] In some embodiments, the 5' UTR comprises or consists of a nucleic acid sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 472.

[0145] In some embodiments, the 5' cap is (m27,3'-O)Gpppp(m2'-O)ApG.

[0146] In some embodiments, the polynucleotide includes one or more modified ribonucleotides. In some embodiments, one or more modified ribonucleotides include pseudouridine.

[0147] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 454. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 454.

[0148] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 455. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 455.

[0149] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 457. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 457.

[0150] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 456. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 456.

[0151] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 461. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 461.

[0152] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 463. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 463.

[0153] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 465. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 465.

[0154] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 462. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 462.

[0155] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 464. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 464.

[0156] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 466. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 466.

[0157] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 459. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 459.

[0158] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 458. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 458.

[0159] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 467. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 467.

[0160] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 460. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 460.

[0161] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 468. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 468.

[0162] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence of SEQ ID NO: 470. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence of SEQ ID NO: 470.

[0163] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 469. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 469.

[0164] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to SEQ ID NO: 471. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to SEQ ID NO: 471.

[0165] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the ribonucleic acid sequence according to any one of SEQ ID NOs: 669 to 864. In some embodiments, the polynucleotide comprises or consists of the ribonucleic acid sequence according to any one of SEQ ID NOs: 669 to 864.

[0166] In some embodiments, the polynucleotide is a non-natural polynucleotide.

[0167] In some embodiments, the polynucleotide is an engineered polynucleotide.

[0168] In some embodiments, the polynucleotide is an isolated polynucleotide.

[0169] In particular among others, the present disclosure provides a composition comprising one or more polynucleotides described herein.

[0170] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 614, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 620.

[0171] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 613, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 619.

[0172] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 617, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 620.

[0173] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 616, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 619.

[0174] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 623, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 620.

[0175] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 622, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 619.

[0176] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 626, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 620.

[0177] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 625, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 619.

[0178] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 635, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 638.

[0179] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 634, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 637.

[0180] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 641, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 638.

[0181] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 640, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 637.

[0182] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 644, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 638.

[0183] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 643, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 637.

[0184] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 647, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 638.

[0185] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 646, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 637.

[0186] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 650, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 638.

[0187] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 649, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 637.

[0188] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 653, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 638.

[0189] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 652, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 637.

[0190] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 635, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence according to SEQ ID NO: 668.

[0191] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 634, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 667.

[0192] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 641, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 668.

[0193] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 640, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 667.

[0194] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 644, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 668.

[0195] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 643, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 667.

[0196] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 647, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 668.

[0197] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 646, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 667.

[0198] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 650, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 668.

[0199] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 649, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences of SEQ ID NO: 667.

[0200] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 653, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 668.

[0201] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 652, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the ribonucleic acid sequences according to SEQ ID NO: 667.

[0202] In some embodiments, the composition comprises or consists of a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 629, and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence according to SEQ ID NO: 632.

[0203] In some embodiments, the composition comprises or consists of a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 628, and a polynucleotide comprising a ribonucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 631.

[0204] In some embodiments, the composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, and one or more polynucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0205] In some embodiments, the composition further comprises lipid nanoparticles, and one or more polynucleotides are encapsulated within the lipid nanoparticles.

[0206] In some embodiments, the lipid nanoparticles target hepatocytes.

[0207] In some embodiments, the lipid nanoparticles target secondary lymphoid organ cells.

[0208] In some embodiments, the lipid nanoparticles target lung cells.

[0209] In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles.

[0210] In some embodiments, each of the lipid nanoparticles comprises a polymer-conjugated lipid, a cationic lipid, and one or more neutral lipids.

[0211] In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid.

[0212] In some embodiments, the polymer-conjugated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0213] In some embodiments, one or more neutral lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0214] In some embodiments, one or more neutral lipids comprise cholesterol.

[0215] In some embodiments, the cationic lipid comprises ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate).

[0216] In some embodiments, each of the lipid nanoparticles comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, DSPC, cholesterol, and ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate).

[0217] In some embodiments, the lipid nanoparticles comprise from about 1 to 2.5 mol% of polymer-conjugated lipid, from 35 to 65 mol% of cationic lipid, based on the total lipid, and one or more neutral lipids are present at from 35 to 65 mol% of the total lipid.

[0218] In some embodiments, the lipid nanoparticles have an average diameter of about 50 to 150 nm.

[0219] The present disclosure also provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a composition provided herein and at least one pharmaceutically acceptable excipient.

[0220] In some embodiments, the medicament comprises a cryoprotectant. In some embodiments, the medicament comprises a buffered aqueous solution.

[0221] In particular, among others, the present disclosure provides a method.

[0222] In some embodiments, the method comprises administering the pharmaceutical composition provided herein to a subject.

[0223] In some embodiments, the pharmaceutical composition provided herein is for use in the treatment of HIV, which comprises administering the pharmaceutical composition to a subject.

[0224] In some embodiments, the pharmaceutical composition provided herein is for use in the prevention of HIV, which comprises administering the pharmaceutical composition to a subject.

[0225] In some embodiments, the method or pharmaceutical composition for use provided herein comprises administering the pharmaceutical composition to a subject, whereby in the subject, the immunoglobulin chain of the antibody agent, the antibody agent, or both are expressed.

[0226] In some embodiments, the immunoglobulin chain of the antibody agent, the antibody agent, or both are expressed in the subject at a titer of at least 1 μg / ml in plasma or serum.

[0227] In some embodiments, the antibody agent exhibits a geometric mean IC50 of less than 0.3 μg / ml for five neutralizing strains against the neutralizing strains of the international reference panel when tested at an antibody agent concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay.

[0228] In some embodiments, the antibody agent has the ability to neutralize one or more HIV strains when tested at an antibody agent concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay.

[0229] In some embodiments, the antibody agent has the ability to neutralize one or more HIV strains at a level within three-fold of the level of an equivalent amount of a recombinant reference antibody.

[0230] In some embodiments, the recombinant reference antibody is an unmodified wild-type IgG antibody that includes the same HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 as the antibody agent.

[0231] In some embodiments, administering the pharmaceutical composition to a subject includes administering one or more doses of the pharmaceutical composition to the subject. In some embodiments, the one or more doses of the pharmaceutical composition are administered to the subject once a week. In some embodiments, the one or more doses of the pharmaceutical composition are administered to the subject twice a week.

[0232] In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered intramuscularly. In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0233] In some embodiments, the subject has or is at risk of developing an HIV infection.

[0234] In some embodiments, the method is a method of treating an HIV infection.

[0235] In some embodiments, the method is a method of preventing an HIV infection.

[0236] Also provided herein is the use of the polynucleotides, compositions, and pharmaceutical compositions described herein.

[0237] In some embodiments, provided is the use of the composition or pharmaceutical composition provided herein for the treatment of HIV in a subject.

[0238] In some embodiments, provided is the use of the composition or pharmaceutical composition provided herein for the prevention of HIV in a subject.

[0239] In some embodiments, the subject has or is at risk of developing HIV infection.

[0240] Furthermore, the present disclosure provides a method of producing an antibody agent. In some embodiments, the method includes administering a composition or a pharmaceutical composition provided herein to a cell such that the cell expresses and secretes the antibody agent as a result.

[0241] In some embodiments, the cell is a hepatocyte.

[0242] In some embodiments, the cell is within the subject.

[0243] In some embodiments, the cell is an ex vivo cell.

[0244] In some embodiments, the antibody agent is produced at a therapeutically appropriate plasma concentration or a therapeutically appropriate serum concentration. In some embodiments, the therapeutically appropriate plasma concentration or the therapeutically appropriate serum concentration is at least 1 μg / ml.

[0245] The present disclosure further provides a method of determining one or more characteristics of an antibody agent expressed from a polynucleotide, a composition or a pharmaceutical composition provided herein. In some embodiments, the polynucleotide, the composition or the pharmaceutical composition provided herein has been introduced into a cell. In some embodiments, the one or more characteristics include: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to the CD4 binding site of HIV; (iii) the effectiveness of the antibody agent to mediate target cell death by antibody-dependent cell-mediated cytotoxicity (ADCC); and (iv) the effectiveness of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC).

[0246] The present disclosure further provides a method comprising contacting a cell with a polynucleotide, composition or pharmaceutical composition provided herein. In some embodiments, the method further comprises detecting an antibody agent produced by the cell.

[0247] In some embodiments, the cell is a hepatocyte.

[0248] In some embodiments, the determining step comprises comparing one or more characteristics of the antibody agent with the one or more characteristics of a reference antibody that specifically binds to the CD4 binding site of HIV.

[0249] In some embodiments, the determining step comprises assessing a protein expression level of the antibody agent that exceeds a threshold level.

[0250] In some embodiments, the threshold level is a level sufficient to induce ADCC.

[0251] In some embodiments, the determining step comprises assessing the binding of the antibody agent to the CD4 binding site of HIV.

[0252] In some embodiments, the determining step comprises assessing the antibody agent at an antibody agent concentration of up to 25 μg / ml in a TZM-bl cell pseudovirus neutralization assay.

[0253] In some embodiments, the cell is present within a subject.

[0254] In some embodiments, the cell is an in vitro cell.

[0255] In some embodiments, the one or more characteristics comprise antibody levels in one or more tissues within a subject.

[0256] The present disclosure also provides a manufacturing method. In some embodiments, the method comprises: (a) determining one or more characteristics of a polynucleotide, composition, or pharmaceutical composition provided herein, wherein the one or more characteristics are (i) the length and / or sequence of the polynucleotide; (ii) the integrity of the polynucleotide; (iii) the presence and / or position of one or more chemical moieties of the polynucleotide; (iv) the degree of expression of the antibody agent when the polynucleotide is introduced into a cell; (v) the stability of the polynucleotide or its composition; (vi) the level of the antibody agent in a biological sample from an organism into which the polynucleotide has been introduced; (vii) the binding specificity of the antibody agent expressed from the polynucleotide, optionally the binding specificity for the CD4 binding site of HIV; (viii) the effectiveness of the antibody agent to mediate target cell death by ADCC; (ix) the effectiveness of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC); (x) the identity and amount / concentration of lipids in the composition; (xi) the particle size of lipid nanoparticles in the composition; (xii) the polydispersity of lipid nanoparticles in the composition; (xiii) the amount / concentration of the polynucleotide in the composition; (xiv) the degree to which the polynucleotide is encapsulated within lipid nanoparticles; (xv) the level of double-stranded RNA; and (xvi) combinations thereof comprising or consisting of said determining; (B) comparing the one or more characteristics of the polynucleotide with the one or more characteristics of a suitable reference standard; and (C)(i) If the comparison shows that the polynucleotide or its composition meets or exceeds the reference standard, designate the polynucleotide or its composition for one or more further steps of manufacture and / or sale, or (C)(ii) Take another measure if the comparison shows that the polynucleotide or its composition does not meet or exceed the reference standard. It includes.

[0257] In some embodiments, the polynucleotide is evaluated, and one or more further steps of step (C)(i) include, at least, formulating the polynucleotide or including it.

[0258] In some embodiments, the composition or pharmaceutical composition is evaluated, and one or more further steps of step (C)(i) include, or include, the release and sale of the composition or pharmaceutical composition.

[0259] Thus, the present disclosure provides a technique that enables the expression of multiple anti-HIV antibodies in a subject, thereby increasing the breadth and efficacy of anti-HIV antibodies present in the subject at one time and reducing the possibility of virus escape.

[0260] The techniques provided are described in more detail herein, including exemplary polynucleotides, compositions containing such polynucleotides, and methods of making and using such polynucleotides.

Brief Description of the Drawings

[0261]

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Mode for Carrying Out the Invention

[0262] Definitions The compounds of the present disclosure include those outlined above and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For the purposes of the present disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Further, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0263] Unless otherwise specified, the structures shown herein are intended to include all conformational isomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each chiral center are contemplated as part of the present disclosure. Accordingly, single stereochemical isomers of the provided compounds, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures are within the scope of the present disclosure. For example, in some cases, the provided compounds exhibit one or more stereoisomers of the compound, and unless otherwise specified, each stereoisomer is represented alone and / or as a mixture. Unless otherwise expressly stated, all tautomeric forms of the provided compounds are within the scope of the present disclosure.

[0264] Unless otherwise specified, the structures shown herein are intended to include compounds that differ only in that they have one or more isotopically enriched atoms. For example, compounds having such structures that include substitution of hydrogen with deuterium or tritium, or substitution of carbon with 13C or 14C enriched carbon are within the scope of the present disclosure.

[0265] About: As used herein with respect to a value, the term "about" refers to a value similar in context to the recited value. In general, one of ordinary skill in the art familiar with the context will fully understand the degree of reasonable variation encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the recited value.

[0266] Agent: As used herein, the term "agent" can refer to a physical entity. In some embodiments, an agent can be characterized by certain features and / or effects. For example, as used herein, the term "therapeutic agent" refers to a physical entity that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect. In some embodiments, an agent can be a compound, molecule, or entity of any chemical type, such as a small molecule, polypeptide, nucleic acid, sugar, lipid, metal, or a combination or complex thereof.

[0267] Aliphatic: The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain having one or more points of attachment to the remainder of the molecule, which is either fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is either fully saturated or contains one or more units of unsaturation but is not aromatic (also referred to herein as "cycloaliphatic"). Unless otherwise specified, an aliphatic group contains from 1 to 12 aliphatic carbon atoms. In some embodiments, an aliphatic group contains from 1 to 6 aliphatic carbon atoms (e.g., C 1-6 ). In some embodiments, an aliphatic group contains from 1 to 5 aliphatic carbon atoms (e.g., C 1-5 ). In other embodiments, an aliphatic group contains from 1 to 4 aliphatic carbon atoms (e.g., C 1-4 ). In still other embodiments, an aliphatic group contains from 1 to 3 aliphatic carbon atoms (e.g., C 1-3 ). In yet other embodiments, an aliphatic group contains from 1 to 2 aliphatic carbon atoms (e.g., C 1-2 ). Suitable aliphatic groups include, but are not limited to, straight-chain or branched substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, and hybrids thereof. Preferred aliphatic groups are C 1-6 alkyl.

[0268] Alkyl: The term "alkyl", used alone or as part of a larger moiety, refers to a saturated, optionally substituted, straight or branched chain hydrocarbon group having from 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms (e.g., C 1-12 , C 1-10 , C 1-8 , C 1-6 , C 1-4 , C 1-3 , or C 1-2 ) unless otherwise specified. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0269] Alkylene: The term "alkylene" refers to a divalent alkyl group. In some embodiments, "alkylene" is a divalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH 2 ) n - where n is a positive integer, e.g., 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are optionally replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups and those described herein. It will be understood that two substituents of an alkylene group may together form a ring system. In certain embodiments, two substituents may together form a 3- to 7-membered ring. Substituents may be on the same or different atoms. The suffix "-en" or "-enyl", when appended to a particular group herein, is intended to refer to the difunctional moiety of said group. For example, when appended to "cyclopropyl", "-en" or "-enyl" becomes "cyclopropylene" or "cyclopropylenyl", referring to a difunctional cyclopropyl group, e.g.,

Chemical formula

[0270] Alkenyl: As used alone or as part of a larger moiety, the term "alkenyl" refers to an optionally substituted straight-chain, branched-chain, or cyclic hydrocarbon group having at least one double bond and (unless otherwise specified) 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0271] Alkynyl: As used alone or as part of a larger moiety, the term "alkynyl" refers to an optionally substituted straight-chain or branched-chain hydrocarbon group having at least one triple bond and (unless otherwise specified) 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0272] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to a compound and / or substance that can be incorporated, has been incorporated, or was incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H 2It has N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid, and in some embodiments, the amino acid is a D-amino acid, and in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether prepared synthetically or obtained from a natural source. In some embodiments, including the carboxy-terminal and / or amino-terminal amino acids in the polypeptide, the amino acid may contain structural modifications compared to the above general structure. For example, in some embodiments, the amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may change the circulating half-life of the polypeptide containing the modified amino acid compared to one containing an unmodified amino acid that is otherwise identical. In some embodiments, such modifications do not change the related action of the polypeptide containing the modified amino acid compared to one containing an unmodified amino acid that is otherwise identical. As will be apparent from the context, the term "amino acid" may be used in some embodiments to refer to a free amino acid and in some embodiments to refer to an amino acid residue of a polypeptide.

[0273] Antibody agent: As used herein, the term "antibody agent" refers to a polypeptide or polypeptide complex that contains immunoglobulin structural elements sufficient to confer specific binding to a particular antigen. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may contain one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody agent may contain one or more sequence elements that have been humanized, primatized, chimerized, etc., as known in the art. In some embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of an antibody in another presentation format. For example, in some embodiments, the antibody agents utilized in accordance with the present disclosure include, but are not limited to, unmodified IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybody®, etc.); CrossMab (e.g., CrossMab CH1-CL , CrossMab CH1-CLcv , bispecific CrossMab with knob-in-hole CH1-CL); antibody fragments, e.g., Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated complementarity-determining regions (CDRs) or combinations thereof; single-chain Fv (scFv); scFv-Fc fusions; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, e.g., IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probody®), small modular immunopharmaceuticals (“SMIP™”); single-chain or tandem diabodies (TandAb®); VHH; anticalin®; nanobody® minibody; BiTE®; ankyrin repeat proteins or DARPIN®; avimer®; DART; TCR-like antibodies; adnectin®; affilin®; transbody®; affibody®; TrimerX®; microprotein; finomer®; centyrin®; and forms selected from KALBITOR®. In some embodiments, chains and / or fragments of such antibodies and fragments can be combined and used, e.g., by combining an scFv-Fc arm with a conventional antibody arm. In some embodiments, the antibody agent is a broad neutralizing antibody agent (e.g., a broad neutralizing antibody (bNAb)). A “broad neutralizing antibody agent” is an antibody agent having the ability to neutralize two or more genetic variants (e.g., lineages) of a virus (e.g., HIV). In some embodiments, the antibody agent can be lacking covalent modifications (e.g., binding to a glycan) that it would have if produced naturally. In some embodiments, the antibody can include covalent modifications (e.g., binding to a glycan, a payload (e.g., a detection moiety, a therapeutic agent moiety, a catalytic moiety, etc.) or other pendant group (e.g., polyethylene glycol, etc.)).In many embodiments, the antibody agent is or comprises a polypeptide that includes in its amino acid sequence one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs). In some embodiments, the antibody agent is or comprises a polypeptide that includes in its amino acid sequence at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the CDR included is substantially identical to the reference CDR in that the sequence is either identical to the reference CDR or contains 1 to 5 amino acid substitutions as compared to the reference CDR. In some embodiments, the CDR included is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDR. In some embodiments, the CDR included is substantially identical to the reference CDR in that it exhibits at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDR. In some embodiments, the CDR included is substantially identical to the reference CDR in that the amino acid sequence of the CDR included is identical to the reference CDR except that at least one amino acid in the CDR included has been deleted, added or substituted as compared to the reference CDR. In some embodiments, the CDR included is substantially identical to the reference CDR in that the amino acid sequence of the CDR included is identical to the reference CDR except that 1 to 5 amino acids in the CDR included have been deleted, added or substituted as compared to the reference CDR. In some embodiments, the CDR included is substantially identical to the reference CDR in that the amino acid sequence of the CDR included is identical to the reference CDR except that at least one amino acid in the CDR included has been substituted as compared to the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that, although 1 to 5 amino acids in the included CDRs are deleted, added or substituted compared to the reference CDRs, the amino acid sequences of the included CDRs are identical to the reference CDRs in other respects. In some embodiments, the antibody agent is or comprises a polypeptide that includes in its amino acid sequence structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or generally homologous to an immunoglobulin binding domain.

[0274] Aryl: The term "aryl" refers to monocyclic and bicyclic ring systems having a total of 6 to 14 ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. In some embodiments, the "aryl" group contains a total of 6 to 12 ring members (e.g., C6-C12). The term "aryl" can be used interchangeably with the term "aryl ring". In certain embodiments, "aryl" refers to an aromatic ring system that includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracyl, etc., and may have one or more substituents. Unless otherwise specified, the "aryl" group is a hydrocarbon. In some embodiments, the "aryl" ring system is an aromatic ring (e.g., phenyl) condensed with a non-aromatic ring (e.g., cycloalkyl). Examples of condensed aryl rings include

Chemical formula

[0275] Related: Two events or entities are "related" to each other when, as the term is used herein, the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic feature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or illness when its presence, level, and / or form correlates (e.g., across an appropriate population) with the incidence, susceptibility, severity, stage, etc. of the disease, disorder, or illness. In some embodiments, two or more entities are physically "related" to each other when they are in proximity to and / or continue to interact directly or indirectly with each other. In some embodiments, two or more entities that are physically related to each other are covalently linked to each other, and in some embodiments, two or more entities that are physically related to each other are not covalently linked but are associated non-covalently, e.g., by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0276] Co - administration: As used herein, the term "co - administration" means the use of a composition described herein (e.g., a pharmaceutical composition) and one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents include at least one polynucleotide encoding another antibody agent (e.g., an anti - HIV antigen antibody agent). The combined use of the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent can be carried out simultaneously or separately (e.g., sequentially in any order). In some embodiments, the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent can be combined in one pharmaceutically acceptable excipient, or they can be in separate excipients and delivered to target cells or administered to a subject at different times. Each of these situations is contemplated to be within the meaning of "co - administration" or "combined use" provided that the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent are delivered or administered sufficiently close in time such that there is at least some temporal overlap in the biological effect(s) on the target cells or the subject being treated produced by each.

[0277] Combination Therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, two or more regimens may be administered simultaneously, and in some embodiments, such regimens may be administered sequentially (e.g., all of the "doses" of the first regimen are administered prior to the administration of any dose of the second regimen), and in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, the administration of combination therapy may involve administering one or more agent(s) or modality(ies) to a subject that is receiving other agent(s) or modality(ies) that are being combined. For the sake of clarity, it should be noted that while in some embodiments two or more agents or their active moieties may be administered together as a combined composition, it is not required that the individual agents be administered together as a single composition (or, in some cases, necessarily simultaneously). In some embodiments, combination therapy comprises a polynucleotide encoding two or more antibody agents (e.g., anti-HIV antibody agents).

[0278] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, etc. that are not necessarily identical to each other, but are sufficiently similar to enable a comparison between them such that a reasonable conclusion can be drawn based on recognized differences or similarities, as would be understood by one of ordinary skill in the art. In some embodiments, an equivalent set of conditions, environments, individuals, or groups is characterized by a plurality of substantially identical features and one or a few different features. One of ordinary skill in the art will understand, in context, what degree of identity is required for two or more such agents, entities, situations, sets of conditions, etc. to be considered equivalent in any given environment. For example, one of ordinary skill in the art will fully understand that sets of environments, individuals, or groups are equivalent to each other if they are characterized by a sufficient number and variety of substantially identical features to warrant the reasonable conclusion that differences in the results obtained or the phenomena observed under or by different sets of environments, individuals, or groups are caused by or represent variations in the modified features.

[0279] Corresponding to: As used herein, the term "corresponding to" refers to the relationship between two or more entities. For example, the term "corresponding to" can be used to specify the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., relative to a suitable reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as corresponding to a residue in a suitable reference polymer. For example, one of ordinary skill in the art will often specify residues in a polypeptide using a standard numbering system based on a reference related polypeptide for the sake of brevity, and as a result, the amino acid that "corresponds to" the residue at position 190 in a particular amino acid chain, for example, may not necessarily be the actual 190th amino acid, but rather corresponds to the residue found at position 190 in the reference polypeptide, and one of ordinary skill in the art will readily recognize a method for identifying the "corresponding" amino acid. For example, one of ordinary skill in the art will be aware of various alignment strategies that can be utilized to identify "corresponding" residues in, for example, polypeptides and / or nucleic acids in accordance with the present disclosure, such as software programs like BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM or SWIPE. Also, one of ordinary skill in the art will recognize that the term "corresponding to" can in some cases be used to represent an event or entity that has a meaningful similarity to another event or entity (e.g., a suitable reference event or entity).By way of example, a gene or protein of one organism may be described as “corresponding to” a gene or protein from another organism, and in some embodiments this description is made to indicate that they perform a similar role or function, and / or exhibit a certain degree of sequence identity or homology, or share certain characteristic sequence elements.

[0280] Cycloaliphatic: As used herein, “cycloaliphatic” refers to a monocyclic C hydrocarbon that is fully saturated or contains one or more units of unsaturation but is not aromatic and has a single point of attachment or more than one point of attachment to the remainder of the molecule. 3-8 Hydrocarbon or bicyclic C 6-10 Hydrocarbon.

[0281] Cycloalkyl: As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system having from about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0282] Derived from: In the context of an amino acid sequence (peptide or polypeptide) “derived from” a specified amino acid sequence (peptide or polypeptide), it refers to a structural analog of the specified amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, an antibody agent utilized according to the present disclosure can include an amino acid sequence (e.g., CDR, variable domain, constant domain, etc.) derived from another antibody, such as a naturally produced antibody.

[0283] To detect: The term "to detect" is used herein in a broad sense to include any suitable means for determining the presence or absence of an entity of interest, or any form of measurement of an entity of interest in a sample. Thus, "to detect" can include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements, or assessments are included, and this includes semi-quantitative ones. Such determinations, measurements, or assessments can be relative or absolute, for example when attempting to detect an entity of interest relative to a control reference. Thus, the term "to quantify" as used in the context of a strategy for quantifying an entity of interest can mean absolute quantification or relative quantification. Absolute quantification can be achieved by correlating the detected level of the entity of interest (e.g., by creating a standard curve) with a known control standard. Alternatively, relative quantification can be accomplished by comparing the detected levels or amounts between two or more different entities of interest to yield a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0284] Dosage regimen: One of ordinary skill in the art will fully understand that the term "dosage regimen" (or "treatment regimen") can typically be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject at intervals over a period of time. In some embodiments, a given therapeutic agent has a recommended dosage regimen that can include more than one dose.

[0285] Encoding: As used herein, the terms "encoding" or "being encoded" refer to the sequence information of a first molecule that induces the production of a second molecule having a defined nucleotide (e.g., polynucleotide) sequence or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process involving a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, cDNA or RNA molecule encodes a polypeptide when a polypeptide is produced intracellularly or within another biological system by transcription and translation of the RNA corresponding to that gene. In some embodiments, the coding region of a polynucleotide encoding a target antigen refers to the coding strand, and the nucleotide sequence of the coding strand is identical to the polynucleotide sequence of such a target antigen. In some embodiments, the coding region of a polynucleotide encoding a target antigen refers to the non-coding strand of such a target antigen, and the non-coding strand can be used as a template for transcription of a gene or cDNA.

[0286] Engineered: Generally, the term "engineered" refers to an artificially manipulated manner. For example, a polynucleotide is considered "engineered" when, in the engineered polynucleotide, two or more sequences that are not naturally ligated together in that order are artificially manipulated to be directly ligated to each other, and / or when a particular residue in the polynucleotide is not naturally occurring and / or is ligated to an entity or moiety that is not naturally ligated by an artificial act.

[0287] Epitope: As used herein, the term "epitope" refers to the portion specifically recognized by the binding component of an immunoglobulin (e.g., an antibody or a receptor). For example, an epitope can be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen is in an appropriate three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen is in such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen is in a different conformation (e.g., linearized). Thus, in some embodiments, the epitope of an antigen can include a continuous or discontinuous portion of the antigen. In some embodiments, the epitope is or includes a T cell epitope. In some embodiments, the epitope can have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0288] Expression: As used herein, the term "expression" of a nucleic acid sequence means the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript, e.g., a polynucleotide provided herein. In some embodiments, the gene product can be a polypeptide. In some embodiments, the expression of a nucleic acid sequence is accompanied by one or more of the following: (1) the production of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) the translation of the RNA into a polypeptide or protein; and / or (4) the post-translational modification of the polypeptide or protein.

[0289] Heteroaliphatic: As used herein, the term "heteroaliphatic" or "heteroaliphatic group" can be linear (i.e., unbranched), branched or cyclic ("heterocyclic"), can be fully saturated or can contain one or more unsaturated units but is not aromatic, and represents an optionally substituted hydrocarbon moiety having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise specified, a heteroaliphatic group contains 1 to 10 carbon atoms, of which 1 to 3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroaliphatic group contains 1 to 4 carbon atoms, of which 1 to 2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, the heteroaliphatic group contains 1 to 3 carbon atoms, of which 1 carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, heteroaliphatic groups having 1 to 10 atoms include the following exemplary groups: -O-CH 3 ,-CH 2 -O-CH 3 ,-O-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH 3 and the like.

[0290] Heteroaryl: The terms "heteroaryl" and "heteroar-" used alone or as part of a larger moiety, such as "heteroalkyl" or "heteroalkoxy", refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl, or 9- to 10-membered bicyclic heteroaryl), having 6, 10 or 14 π electrons shared in a cyclic arrangement, and having 1 to 5 heteroatoms in addition to carbon atoms. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzisoxazolyl. The terms "heteroaryl" and "heteroar-" as used herein also include groups in which the heteroaromatic ring is fused to one or more aryl, cycloaliphatic or heterocyclyl rings and the radical or point of attachment is on the heteroaromatic ring (i.e., bicyclic heteroaryl rings having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzisoxazolyl.The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", although each of these terms includes optionally substituted rings.

[0291] Heteroatom: As used herein, the term "heteroatom" refers to nitrogen, oxygen or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen.

[0292] Heterocyclic ring: As used herein, the terms "heterocyclic ring", "heterocyclyl", "heterocyclic radical" and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 8-membered monocyclic, 6- to 10-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated and has one or more, for example 1 to 4, of the heteroatoms as defined above in addition to carbon atoms. The term "nitrogen", when used with respect to the ring atoms of a heterocyclic ring, includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen may be N (as in the case of 3,4-dihydro-2H-pyrrolyl), NH (as in the case of pyrrolidinyl), or NR +It can be (as in the case of N-substituted pyrrolidinyl). The heterocyclic ring may be attached to the pendant group at any heteroatom or carbon atom so as to provide a stable structure, and any of the ring atoms may optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiomorpholinyl. The heterocyclyl group can be monocyclic, bicyclic, tricyclic or polycyclic, preferably monocyclic, bicyclic or tricyclic, more preferably monocyclic or bicyclic. The bicyclic heterocyclic ring also includes groups in which a heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. The bicyclic heterocyclic ring may also be a spirocyclic ring system (e.g., a 7- to 11-membered spirocyclic fused heterocyclic ring having one or more heteroatoms (e.g., 1, 2, 3 or 4 heteroatoms) defined above in addition to carbon atoms). The bicyclic heterocyclic ring may also be a bridged ring system (e.g., a 7- to 11-membered bridged heterocyclic ring having 1, 2 or 3 bridging atoms).

[0293] Identity: As used herein, the terms "identity" or "identical" mean the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be identical to each other when their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be identical to each other when their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). For example, as is well known to those of skill in the art, certain amino acids are typically similar to each other as "hydrophobic" or "hydrophilic" amino acids and / or are classified as having "polar" or "non-bonding" side chains. Substituting one amino acid for another of the same type can often be considered a "conservative" substitution.

[0294] Identity: As used herein, the term "identity" means the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "substantially identical" to each other if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. The percent identity between two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Thereafter, the nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which is incorporated into the ALIGN program (version 2.0).In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program use the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package that uses the NWSgapdna.CMP matrix.

[0295] Increased, induced, or decreased: As used herein, these terms or grammatically equivalent comparative terms refer to a value relative to a comparable reference measurement. For example, in some embodiments, an evaluation value provided using a provided composition (e.g., a pharmaceutical composition) may be "increased" compared to an evaluation value obtained using a comparable reference composition. Alternatively, or additionally, in some embodiments, an evaluation value provided in a subject is obtained in the same subject under different conditions (e.g., before or after an event, or in the presence or absence of an event, such as the administration of a composition (e.g., a pharmaceutical composition) described herein) or in a different comparable subject (e.g., a comparable subject that is different from the subject of interest in that it was previously exposed to a condition, such as the absence of the administration of a composition (e.g., a pharmaceutical composition) described herein) may be "increased" compared to an evaluation value obtained. In some embodiments, the comparative term means that there is a statistically significant difference (e.g., of sufficient magnitude and / or preponderance to result in statistical significance). One of ordinary skill in the art will recognize or be able to readily determine the degree of difference and / or preponderance necessary or sufficient to result in such statistical significance in a given context. In some embodiments, the term "decreased" or a synonym means that the level of the evaluation value is reduced by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or more compared to a comparable reference. In some embodiments, the term "decreased" or a synonym means a complete or substantially complete inhibition, i.e., a reduction to zero or a reduction substantially to zero. In some embodiments, the terms "increased" or "induced" mean that the level of the evaluation value is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500% or more compared to a comparable reference.

[0296] In order: As used herein with respect to a polynucleotide or polyribonucleotide, "in order" refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with respect to a polypeptide, "in order" refers to the order of features moving from the most N-terminal feature to the most C-terminal feature along the polypeptide. "In order" does not mean that there cannot be additional features between the recited features. For example, if features A, B, and C of a polynucleotide are described herein as being "in order feature A, feature B, and feature C", this description does not exclude, for example, the possibility that feature D is located between feature A and feature B.

[0297] Ionizable: The term "ionizable" refers to a compound or group or atom that becomes charged at a particular pH. In the context of an ionizable amino lipid, such a lipid or its functional group or atom is positively charged at a particular pH. In some embodiments, the ionizable amino lipid is positively charged at acidic pH. In some embodiments, the ionizable amino lipid is predominantly neutral at physiological pH values, e.g., about 7.0 - 7.4 in some embodiments, but becomes positively charged at lower pH values. In some embodiments, the ionizable amino lipid can have a pKa in the range of about 5 to about 7.

[0298] Isolated: The term "isolated" means modified or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting materials is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as a host cell.

[0299] Lipids: As used herein, the terms “lipid” and “lipid-like substance” are broadly defined as molecules that include one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules that contain both hydrophobic and hydrophilic moieties are often also referred to as amphiphilic substances.

[0300] RNA lipid nanoparticles: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle that includes at least one lipid and an RNA molecule(s), such as one or more polynucleotides provided herein. In some embodiments, the RNA lipid nanoparticle includes at least one cationic amino lipid. In some embodiments, the RNA lipid nanoparticle includes at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., a PEG-conjugated lipid). In various embodiments, the RNA lipid nanoparticles described herein can have an average particle size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, the RNA lipid nanoparticle can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average particle size of the lipid nanoparticle is determined by measuring the average particle diameter. In some embodiments, the RNA lipid nanoparticle can be prepared by mixing the lipid with the RNA molecule(s) described herein.

[0301] Neutralization: As used herein, the term "neutralization" refers to the event in which a binding agent, such as an antibody, binds to the biological active site of a virus, such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term "neutralization" refers to the event in which the binding agent eliminates or significantly reduces the ability to infect cells.

[0302] Nucleic acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, the nucleic acid is or contains DNA. In some embodiments, the nucleic acid is or contains RNA. In some embodiments, the nucleic acid is or contains peptide nucleic acid (PNA). In some embodiments, the nucleic acid is or contains single-stranded nucleic acid. In some embodiments, the nucleic acid is or contains double-stranded nucleic acid. In some embodiments, the nucleic acid contains both single-stranded and double-stranded portions. In some embodiments, the nucleic acid contains a backbone that includes one or more phosphodiester bonds. In some embodiments, the nucleic acid contains a backbone that includes both phosphodiester bonds and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may contain a backbone that includes one or more phosphorothioate bonds or 5'-N-phosphoramidite bonds, and / or one or more peptide bonds such as those found in "peptide nucleic acid". In some embodiments, the nucleic acid contains one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid contains one or more or all non-natural residues. In some embodiments, the non-natural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).In some embodiments, the unnatural residue, as contrasted with the sugars in natural residues, comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500 or 20,000 residues or nucleotides in length, or more.

[0303] Pharmaceutically effective amount: The term "pharmaceutically effective amount" or "therapeutically effective amount" refers, alone or in conjunction with further dosages, to an amount that produces a desired response or desired effect. In the treatment of a particular disease (e.g., HIV), in some embodiments the desired response relates to inhibition of the course of the disease (e.g., HIV). In some embodiments, such inhibition may include slowing the progression of the disease (e.g., HIV) and / or arresting or reversing the progression of the disease (e.g., HIV). In some embodiments, the desired response in the treatment of a disease (e.g., HIV) may be or may include delaying or preventing the onset of the disease (e.g., HIV) or a disorder (e.g., an HIV-related disorder). The effective amount of a composition (e.g., a pharmaceutical composition) described herein is determined, for example, by the disease (e.g., HIV) or disorder (e.g., an HIV-related disorder) being treated, the severity of such disease (e.g., HIV) or disorder (e.g., an HIV-related disorder), the individual parameters of the patient, such as age, physiological state, size and weight, the duration of the treatment, the type of concomitant therapy (if any), the particular route of administration, and like factors. Accordingly, the dosage of a composition (e.g., a pharmaceutical composition) described herein can be determined by a variety of such parameters. If the patient's response is inadequate at the initial dosage, a higher dosage (or, in effect, a higher dosage achieved by a different, more local route of administration) may be used.

[0304] Polypeptide: As used herein, the term "polypeptide" refers to a polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an engineered amino acid sequence in that it is designed and / or produced by human intervention. In some embodiments, the polypeptide can comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide can comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide can comprise only D-amino acids. In some embodiments, the polypeptide can comprise only L-amino acids. In some embodiments, the polypeptide can comprise one or more pendant groups or other modifications, such as modifying or attaching one or more amino acid side chains, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications include acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, the polypeptide can be cyclic and / or can comprise a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not comprise any cyclic moieties. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can be or can comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, in which case the term is used herein to refer to a polypeptide that can be considered a member of the same class or family of polypeptides that share the relevant activity or structure.For each such class, exemplary polypeptides within the class for which the amino acid sequence and / or function are known are provided herein and / or will be known to those of skill in the art. In some embodiments, such exemplary polypeptides are reference polypeptides for a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit a relatively high degree of sequence homology or identity with the reference polypeptide of that class, in some embodiments with all polypeptides within that class, share a common sequence motif (e.g., a characteristic sequence element), and / or share a common activity (in some embodiments, at an equivalent level or within a specified range). For example, in some embodiments, the polypeptide of a member exhibits an overall degree of sequence homology or identity of at least about 30-40%, often greater than about 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or more with the reference polypeptide, and / or often exhibits a very high sequence identity of greater than 90% or, in some cases, greater than 95%, 96%, 97%, 98%, or 99% in at least one region (e.g., a conserved region that can be or can include a characteristic sequence element in some embodiments). Such conserved regions typically include at least 3-4, often more than 35 amino acids, and in some embodiments, the conserved region includes at least one range of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more contiguous amino acids. In some embodiments, the related polypeptide can include or consist of a fragment of the parent polypeptide.

[0305] Prevent: As used herein, the term "prevent" or "preventing" when used in connection with the occurrence of a disease, disorder, and / or illness means reducing the risk of onset of the disease, disorder, and / or illness and / or delaying the onset of one or more features or symptoms of the disease, disorder, or illness. Prevention can be considered complete when the onset of the disease, disorder, or illness has been delayed over a predetermined period of time.

[0306] Reference: As used herein, the term "reference" refers to a standard or control to which comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is optionally a past reference or control recorded on a tangible medium. Typically, as would be understood by one of ordinary skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those of the subject being evaluated. One of ordinary skill in the art will fully understand in what cases there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control considered.

[0307] Ribonucleic acid (RNA) or polynucleotide: As used herein, the terms "ribonucleic acid", "RNA" or "polynucleotide" refer to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA contains both single-stranded and double-stranded portions. In some embodiments, the RNA may contain the backbone structure described in the above definition of "nucleic acid / polynucleotide". The RNA can be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments, the RNA is mRNA and the RNA typically contains a poly(A) region at its 3' end. In some embodiments where the RNA is mRNA, the RNA typically contains, at its 5' end, a cap structure recognized in the art for recognition and for binding the mRNA to ribosomes to initiate translation. In some embodiments, the RNA is synthetic RNA. Examples of synthetic RNA include RNA synthesized in vitro (e.g., by enzymatic synthesis methods and / or chemical synthesis methods).

[0308] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can contain one or more modifications, including, but not limited to, for example, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (e.g., conjugation, inverted linkage, etc.), (b) base modifications, such as modified bases, stabilized bases, destabilized bases, or bases that base pair with an extended repertoire of partners, or replacement by conjugated bases, (c) sugar modifications (e.g., at the 2'- or 4'-position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modifications or replacements of the phosphodiester bond. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0309] Risk: As will be understood from the context, the "risk" of a disease, disorder, and / or illness refers to the likelihood that a particular individual will develop the disease, disorder, and / or illness. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is expressed as a relative risk to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of a disease, disorder, illness, and / or event. In some embodiments, the reference sample or group of reference samples is obtained from an individual equivalent to a particular individual. In some embodiments, the risk can reflect one or more genetic attributes that can confer upon an individual a predisposition to the development (or non-development) of a particular disease, disorder, and / or condition. In some embodiments, the risk can reflect one or more epigenetic events or attributes, and / or one or more events or attributes due to lifestyle or environment.

[0310] Selective or specific: As used herein with respect to an agent having activity, it will be understood by those skilled in the art that the terms “selective” or “specific” mean that the agent discriminates between potential target entities, conditions or cells. For example, in some embodiments, an agent is said to “specifically” bind to its target if the agent preferentially binds to its target in the presence of one or more competing alternative targets. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the target binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binding moiety.

[0311] Substituted or optionally substituted: As described herein, the compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether or not preceded by the term “optionally,” means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are explicitly or implicitly present in the structure (e.g.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0312] Suitable monovalent substituents on a replaceable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH 2 ) 0-4 R°; -(CH 2 ) 0-4 OR°; -O(CH 2 ) 0-4 R°; -O-(CH 2 ) 0-4 C(O)OR°; -(CH 2 ) 0-4 CH(OR°) 2 ; -(CH 2 ) 0-4 SR°; -(CH 2 ) 0-4 Ph; -(CH 2 ) 0-4 O(CH 2 ) 0-1 Ph; -CH=CHPh optionally substituted with R°; -(CH 2 ) 0-4 O(CH 2 ) 0-1 -pyridyl; -NO2 ; -CN; -N 3 ; -(CH 2 ) 0-4 N(R°) 2 ; -(CH 2 ) 0-4 N(R°)C(O)R°; -N(R°)C(S)R°; -(CH 2 ) 0-4 N(R°)C(O)NR° 2 ; -N(R°)C(S)NR° 2 ; -(CH 2 ) 0-4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR° 2 ; -N(R°)N(R°)C(O)OR°; -(CH 2 ) 0-4 C(O)R°; C(S)R°; -(CH 2 ) 0-4 C(O)OR°; -(CH 2 ) 0-4 C(O)SR°; -(CH 2 ) 0-4 C(O)OSiR° 3 ; -(CH 2 ) 0-4 OC(O)R°; -OC(O)(CH 2 ) 0-4 SR°; -(CH 2 ) 0-4 SC(O)R°; -(CH 2 ) 0-4 C(O)NR° 2 ; -C(S)NR° 2 ; -C(S)SR°; -SC(S)SR°、-(CH 2 ) 0-4 OC(O)NR° 2 ; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH 2 C(O)R°; -C(NOR°)R°; -(CH 2 ) 0-4 SSR°; -(CH 2 ) 0-4 S(O) 2 R°; -(CH 2 ) 0-4 S(O) 2 OR°; -(CH 2 )0-4 OS(O) 2 R°;-S(O) 2 NR° 2 ;-(CH 2 ) 0-4 S(O)R°;-N(R°)S(O) 2 NR° 2 ;-N(R°)S(O) 2 R°;-N(OR°)R°;-C(NH)NR° 2 ;-P(O) 2 R°;-P(O)R° 2 ;-OP(O)R° 2 ;-OP(O)(OR°) 2 ;SiR° 3 ;-(C 1-4 linear or branched alkylene)O-N(R°) 2 ; or -(C 1-4 linear or branched alkylene)C(O)O-N(R°) 2 wherein each R° may be substituted as defined below and is independently hydrogen, C 1-6 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, -CH 2 -(5- to 6-membered heteroaryl ring), or may independently be a 3- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms selected from nitrogen, oxygen or sulfur, or, regardless of the above definition, two independently occurring R° may together with the intervening atom(s) form an independently 3- to 12-membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms selected from nitrogen, oxygen or sulfur, and these may be substituted as defined below.

[0313] Preferred monovalent substituents on R° (or the ring formed by combining two independently occurring R° with the intervening atom(s)) are independently halogen, -(CH 2 ) 0-2 R | 、-(haloR | )、-(CH 2 ) 0-2OH, -(CH 2 ) 0-2 OR | , -(CH 2 ) 0-2 CH(OR | ) 2 , -O(haloR | ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R | , -(CH 2 ) 0-2 C(O)OH, -(CH 2 ) 0-2 C(O)OR | , -(CH 2 ) 0-2 SR | , -(CH 2 ) 0-2 , -(CH 2 ) 0-2 NH 2 , -(CH 2 ) 0-2 NHR | , -(CH 2 ) 0-2 NR | 2 , -NO 2 , -SiR | 3 , -OSiR | 3 , -C(O)SR | , -(C 1-4 linear or branched alkylene)C(O)OR | , or -SSR | wherein each R | is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens and is independently selected from C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or independently selected from 3- to 6-membered saturated, partially unsaturated or aryl rings having 0 to 4 heteroatoms selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on the saturated carbon atoms of R° include =O and =S.

[0314] Suitable divalent substituents on a saturated carbon atom of a "optionally substituted" radical include the following: =O ("oxo"), =S, =NNR * 2 , =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O) 2 R * , =NR * , =NOR * , -O(C(R * 2 )) 2-3 O-, or -S(C(R * 2 )) 2-3 S-, where each R that appears independently in the formula * is hydrogen, C 1-6 aliphatic optionally substituted as defined below, or unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl rings having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bonded to vicinal substitutable carbons of a "optionally substituted" radical include -O(CR * 2 ) 2-3 O-, where each R that appears independently in the formula * is hydrogen, C 1-6 aliphatic optionally substituted as defined below, or unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl rings having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0315] R * Suitable substituents on the aliphatic group of | include halogen, -R | , -(haloR | ), -OH, -OR | , -O(haloR | ), -CN, -C(O)OH, -C(O)OR 2 , -NH | , -NHR | 2 , or -NO 2 , where in the formula, each R |is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently, C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or is a 3- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0316] Suitable substituents on the nitrogen of the "optionally substituted" group that can be substituted include -R † , -NR † 2 , -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH 2 C(O)R † , -S(O) 2 R † , -S(O) 2 NR † 2 , -C(S)NR † 2 , -C(NH)NR † 2 , or -N(R † )S(O) 2 R † may be mentioned; wherein each R † is independently hydrogen, C 1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, regardless of the above definition, two independently occurring R † together with the intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0317] R † Suitable substituents on the aliphatic group of are independently halogen, -R| 、 -(halo R | )、 -OH、 -OR | 、 -O(halo R | )、 -CN、 -C(O)OH、 -C(O)OR | 、 -NH 2 、 -NHR | 、 -NR | 2 、 or -NO 2 ; wherein each R | is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently is C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or independently is a 3- to 6-membered saturated, partially unsaturated or aryl ring having from 0 to 4 heteroatoms selected from nitrogen, oxygen or sulfur.

[0318] Subject: As used herein, the term "subject" refers to an organism to which a composition described herein is administered for purposes such as, for example, experimentation, diagnosis, prevention and / or treatment. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, household pets, etc.), and humans. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject is susceptible to a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject exhibits no symptoms or characteristics of a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject is a person having one or more characteristics characteristic of susceptibility or risk to a disease, disorder or illness (e.g., HIV, HIV-related diseases, etc.). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual on whom and / or who has undergone diagnosis and / or treatment.

[0319] Suffering from: An individual suffering from a disease, disorder and / or illness (e.g., HIV, HIV-related diseases, etc.) is diagnosed with the disease, disorder and / or illness and / or exhibits one or more symptoms thereof.

[0320] Prone to: Diseases, disorders, and / or illnesses (e.g., HIV, HIV-related illnesses, etc.) An individual who is "prone to" a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) is an individual with a higher risk of developing the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) compared to the general population. In some embodiments, an individual who is prone to a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) may not be diagnosed with the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, an individual who is prone to a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) may exhibit symptoms of the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, an individual who is prone to a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) may not exhibit symptoms of the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, an individual who is prone to a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) will develop the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, an individual who is prone to a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) will not develop the disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.).

[0321] Therapy: The term "therapy" means the administration or delivery of an agent or intervention that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect (e.g., demonstrated to have a high statistical likelihood of having such an effect when administered to the relevant population). In some embodiments, a therapeutic agent is any substance that can be used to alleviate, improve, reduce, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, reduce, suppress, present, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or illness.

[0322] Treat: As used herein, the terms "treat", "treatment" or "treating" refer to any method that can be used to partially or completely alleviate, improve, reduce, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). Treatment may be administered to a subject who does not exhibit symptoms of a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some embodiments, treatment may be administered to a subject who exhibits only the initial symptoms of a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.) for the purpose of, for example, reducing the risk of developing a medical condition associated with the disease, disorder, and / or illness. In some embodiments, treatment may be administered to a subject at a later stage of a disease, disorder, and / or illness (e.g., HIV, HIV-related illnesses, etc.).

[0323] Detailed Description of Particular Embodiments I. Human Immunodeficiency Virus (HIV) Human immunodeficiency virus (HIV) is a lentivirus within the Retroviridae family. Mature HIV particles are generally circular and approximately 100 nm in diameter. It consists of a core, a capsid, and an envelope composed of two identical single-stranded RNA molecules (from the innermost to the outermost) (Figure 1B) (Musumeci et al., Molecules 20.9 (2015): 17511-17532, incorporated herein by reference). The envelope consists of a lipid bilayer and Env proteins. These Env proteins exist as trimers of gp120 surface proteins tethered to the envelope membrane via the gp41 transmembrane protein. The viral capsid surrounded by the envelope contains a symmetric outer capsid membrane, which is composed of the matrix protein p17. Inside the outer capsid membrane, there is a conical capsid composed of the inner capsid protein p24. The inner capsid is bound to the outer capsid membrane at its tapered cone. The inner capsid contains viral RNA (two identical copies) and viral enzymes, namely reverse transcriptase, integrase, and protease. Also contained within the viral particle are oligopeptides generated by the proteolytic processing of the Gag and Gag / Pol precursor proteins p55 and p160 that occur during viral particle maturation (GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, incorporated herein by reference).

[0324] There are two main types of HIV, namely HIV-1 and HIV-2. HIV-1 is the most common type of HIV, accounting for 95% of all infections worldwide. HIV-2 is relatively rare and less infectious. HIV-2 is mainly concentrated in West Africa and surrounding countries.

[0325] HIV-1 and HIV-2 have many similarities, including their intracellular replication pathways, modes of transmission, and clinical implications leading to acquired immunodeficiency syndrome (AIDS). However, due to its lower infectivity, HIV-2 is less likely to progress to AIDS. For this reason, individuals infected with HIV-2 generally do not show long-term disease progression, while patients infected with HIV-1 progress more rapidly and tend to develop AIDS.

[0326] However, once progression begins, the pathological processes of both viruses are nearly identical. One difference is that HIV-2 has been found to progress at a higher CD4 count. Additionally, HIV-2 infection is characterized by a lower viral load, exceeding 10,000 copies / mL compared to millions of copies / mL in the case of HIV-1. In the case of HIV-2 infection, the immune response of the subject tends to be more defensive, thus slowing the progression of the disease.

[0327] HIV-1 and HIV-2 are further classified into groups and subtypes. HIV-1 is classified into the major group or M group, the outlier group or O group, and the non-M / O group or N group. The most common group is group M, which is the main cause of the global HIV epidemic. The other groups are relatively rare and are found in limited geographies including Gabon, Cameroon, and Equatorial Guinea.

[0328] Group M is further classified into genetically distinct subtypes: A, B, C, D, F, G, H, J, and K. Some of these subtypes combine to form hybrid viruses called "recombinant epidemic strains." Subtype B accounts for 12% of HIV infections worldwide. Subtype B is the dominant HIV-1 subtype found in the United States, Australasia, and Western Europe. Accordingly, most clinical research on HIV to date has focused on these populations.

[0329] Subtype C accounts for almost 50% of all HIV-infected individuals, but there have been fewer studies focusing on this subtype. Subtype C is commonly found in countries in southern Africa, where the HIV incidence is very high. In Cameroon and the Democratic Republic of the Congo, the regions where HIV-1 originated, there are a variety of HIV-1 subtypes. However, the global subtype distribution pattern is currently changing due to the mixing and movement of people.

[0330] To date, approximately eight subtypes of HIV-2 have been identified. The two major subtypes of HIV-2 that are considered to be epidemic are A and B. Infections with HIV-2 group A are mainly seen in West Africa, but a few cases have been reported in Brazil, Europe, the United States, and India. Infections with HIV-2 group B are only seen in West Africa.

[0331] Since the subtypes of HIV can be geographically diverse, an ideal therapeutic agent is one that can neutralize more than one subtype of HIV, and more preferably multiple strains. As further discussed below, anti-HIV antibodies have been developed that have the ability to bind to HIV virions and neutralize them, at least temporarily. Nevertheless, problems with such anti-HIV antibodies still remain, including issues related to administration, antibody persistence in vivo, and viral escape. The polynucleotides and compositions of the present disclosure address these issues, as described herein.

[0332] A. The HIV Genome HIV contains two identical copies of single-stranded DNA encoding its genome. When the virus integrates into a host cell, reverse transcription from viral RNA to double-stranded DNA occurs, which leads to the degradation of RNA and the integration of double-stranded DNA or proviral DNA into the host genome. Adjacent to both ends of the HIV genome is an LTR (long terminal repeat) region that includes a 5’ LTR encoding a transcriptional promoter. The RNA genome is 9,749 nucleotides and contains a 5’ cap, a 3’ poly(A) tail, and several open reading frames ORFs (see Wain-Hobson et al., Cell 40(1):9-17, 1985, which is incorporated herein by reference).

[0333] The HIV genome contains the following genes: gag, pol, vif, vpr, tat, rev, vpu, env, and nef (see Figure 1A). The proteins encoded by gag, pol, and env are viral structural proteins. The proteins encoded by tat and ref are essential regulatory proteins. The proteins encoded by nef, vpr, vif, and vpu are accessory regulatory proteins. The gag gene encodes the P555Gag precursor protein of a protein containing an outer core membrane (p17), a capsid protein (p24), a nucleoprotein (p7), Pr55Gag, and a p6 protein. The protein p24 forms a conical capsid, and the protein p17 forms an inner membrane layer. The protein p6 is involved in the release of viral particles.

[0334] The pol gene encodes the Pr160GagPol precursor protein, the protease enzyme p10, the reverse transcriptase (p51), and the RNase H (p15), or both of them together as the p66 protein, and the integrase p32. Pr160GagPol is the precursor of the viral enzymes p10, p51 and p15. Proteolytic cleavage of Gag (Pr55) and Gag-Pol (Pr160GagPol) results in the protease p10. The protein p51 reverse transcriptase is responsible for the transcription of HIV RNA into proviral DNA. The protein p55 (RNase H) functions to degrade the viral RNA in the viral RNA / DNA complex when proviral DNA is generated. The protein p32 integrase functions in the integration of proviral DNA into the host cell genome.

[0335] The env gene encodes the precursor protein PrGp160 of the two envelope glycoproteins gp120 (surface protein) and gp41 (transmembrane protein). The proteins gp120 and gp41 are generated by proteolytic cleavage of the precursor protein PrGp160. The protein gp120 functions in the binding of the virus to the target host cell. The protein gp41 functions to tether gp120 to the viral membrane and to fuse the viral membrane with the target cell membrane.

[0336] The gene tat encodes the Tat protein p14 (trans-activator of transcription protein), which activates the transcription of viral genes. The rev gene encodes the Rev protein p19 (RNA splicing regulator), which regulates the export of mRNA (both unspliced and partially spliced). The nef gene encodes the Nef protein p27 (negative regulator), which functions in the replication of HIV and enhances the infectivity of the virus in host cells. The protein p27 also functions to downregulate CD4 and HLA on the target cells. The vif gene encodes the Vif protein p23 (viral infectivity factor), which functions in the production of the virus in host cells. The gene vpr encodes the Vpr protein p15 (viral protein r). This protein interacts with the p6 protein and promotes the infectivity of the virus in host cells. The gene vpu encodes the Vpu protein p16 (virus protein unique), which enables the efficient release of viral particles and controls the degradation of CD4 on the target cells. The protein p16 also controls intracellular signal transduction. The gene vpx encodes the Vpx protein p15 (viral protein x), which functions to interact with the p6 protein and is important in the early stages of virus replication. The gene tev encodes the Tat / Rev protein p26, which is a fusion protein that regulates the Tat and Rev proteins (GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, which is incorporated herein by reference).

[0337] B. Life cycle The life cycle of HIV includes the entry of HIV virions into target host cells, reverse transcription of the viral genome, integration into the host genome, and protein maturation. To initiate infection, HIV particles contact target host cells. The surface glycoprotein env gp120 of mature HIV particles binds to the CD4 receptor on target host cells, which in turn causes further binding of gp120 to a co-receptor, namely chemokine receptor 5 (CCR5) or chemokine receptor 4 (CXCR4, fusin). Binding of gp120 to CD4 and the co-receptor causes a conformational change in gp120, as a result of which gp41 is presented on the viral membrane and can fuse with the plasma membrane of the target host cell. Subsequently, the viral capsid enters the cytoplasm of the host cell. The capsid is taken up by endosomes and releases its contents, namely viral RNA. Entry and release of the virus into the target host cell subject the virus to reverse transcription, whereby the viral RNA is reverse transcribed into single-stranded cDNA. Subsequently, the RNA strand is degraded by RNase H and the single-stranded cDNA is converted into double-stranded DNA by the DNA-dependent DNA polymerase activity of the reverse transcriptase enzyme.

[0338] The double-stranded DNA, or proviral DNA, forms a complex with integrase, is transported into the nucleus of the host cell, and randomly inserts itself into the host cell genome. The proviral genome is replicated once it is integrated into the genome. The proviral genome can be replicated along with the host cell genome as part of cell division or by using mechanisms it has on its own. For example, the LTR promoter creates binding sites for cellular DNA-dependent RNA polymerase and transcription factors to initiate transcription. Transcription of proviral DNA is accelerated by the Tat protein.

[0339] The processes of entry into the target host cell, reverse transcription, integration, and protein maturation can be completed in less than 24 hours, and progeny virus particles have been detected within 12 hours of infection. The first progeny virus particles after infection can be released from infected cells at about 24 hours post-infection. Infected T cells are typically eliminated by the immune system (e.g., by cytotoxic T cells) at a rate of 2 - 4 days. Since HIV-infected T cells are destroyed and there is a limit to T cell production, helper T cells will decrease. Proteins nef and tat also inhibit the maturation and replacement of helper T cells. As a result, HIV infection will eventually lead to immunodeficiency (GAC, Transfusion Medicine Hemotherapy, 43:203 - 222, 2016, which is incorporated herein by reference).

[0340] C. Transmission and Pathology HIV enters the body through intact mucous membranes, broken skin, or by parenteral inoculation. HIV is most commonly transmitted through sexual intercourse. Once infected, HIV can be detected systemically with a lag of about 10 - 14 days, and transmission via blood or transplanted organs is possible about 5 - 6 days after infection. Clinical symptoms typically appear 3 - 6 weeks after infection and may include fever, lymphadenopathy, malaise, rash, gastrointestinal symptoms, acute neuropathy, myalgia, and / or discomfort. However, during this acute phase, some individuals are asymptomatic. These symptoms of acute or primary infection may last 2 - 6 weeks. Then, after this initial symptomatic period, there is typically an asymptomatic period or a period with occasional symptoms, which may last for several years.

[0341] Untreated HIV infection causes progressive CD4+ T cell loss, which can lead to a series of immunological abnormalities and an increased risk of infectious and oncological complications. In addition, HIV infection is necessarily accompanied by cardiovascular disease, bone disease, kidney and liver dysfunction, and several other common pathological conditions.

[0342] Antiretroviral therapy (ART) has been developed to treat HIV infection, but ART can only prevent new cells from being infected. That is, ART cannot eliminate infection if the cells already contain viral DNA integrated into their genome. Furthermore, HIV can establish latent infections in CD4+ T cells that can be maintained indefinitely, and some have the ability to self-renew. Once integrated into the cell's genome, HIV may resume replication. (See Deeks et al., Nature Reviews 1.1 2015, and GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, which are incorporated herein by reference).

[0343] D. Treatment Strategies The development of therapeutics targeting HIV faces many challenges. One difficult factor is the virus's heterogeneity. HIV can be divided into at least two major types (HIV-1 found worldwide and HIV-2 found mainly in West Africa), but HIV-1 is further subdivided into three subgroups (M, N, O, and P), and M is further subdivided into subtypes A-L. Subtypes can recombine when co-infected, resulting in even more recombinant subtypes.

[0344] Another difficult factor is the high mutation rate of HIV in vivo. Recent studies have quantified the natural mutation rate across the entire HIV-1 genome in DNA sequences from peripheral blood mononuclear cells, revealing an extremely high mutation rate of (4.1±1.7)×10 -3 per base per cell, which is the highest mutation rate reported for any biological entity (see Cuevas et al., PloS Biol 2015, which is incorporated herein by reference). Therefore, the ability to identify and develop therapeutics targeting epitopes conserved across multiple groups and subtypes of the continuously mutating HIV sequences is extremely difficult, and the virus has a unique ability to evade the immune system.

[0345] In addition to its high mutation rate, HIV presents the immune system with other daunting challenges that make treatment extremely difficult. Despite the fact that treatment targets on HIV include the HIV envelope protein (HIV Env), HIV Env is highly glycosylated, and thus the Env site is shielded from therapeutic agents by the glycans present. In addition, Env glycans are host-derived and can be highly heterogeneous.

[0346] Recent treatment strategies involve the use of broadly neutralizing antibodies (bNAbs), which are antibodies that can neutralize diverse international HIV isolates. Such antibodies have been identified from HIV-infected individuals classified as "Elite Neutralizers," who constitute less than 10% of HIV patients (Burton and Hangartner, Ann. Rev. Immunol. 2016, which is incorporated herein by reference). Such antibodies provide insights into potential target epitopes and the structure of therapeutic agents. Other advances that have helped in the progress of therapeutic agents include the generation of stable HIV Env spike trimers (Sanders and Moore, Immunol. Rev. 2017, which is incorporated herein by reference), and the high-resolution characterization of their structure (Ward and Wilson, Immunol. Rev. 2017, which is incorporated herein by reference). Examples of potential target Env sites include the apex site, the high-mannose portion of the gp120 region, the gp120-gp41 interface region, the gp41 membrane-proximal region (MPER), and the CD4 binding site (see Figure 2, from McCoy and Burton, Immunol Rev. 275.1 11-20 2017, which is incorporated herein by reference). Each of these sites faces unique challenges as therapeutic targets for bNAbs. For example, bNAbs targeting the gp41-gp120 interface must be able to bind to complex and heterogeneous glycans. BNAbs targeting the CD4 binding site of the Env protein have been found to exhibit a high level of somatic hypermutation.

[0347] Nevertheless, among these sites, CD4b is of particular interest because CD4 serves as the primary receptor for viral entry. Certain CD4b bNAbs have been characterized by their use of immunoglobulin heavy chain gene segment IGVH1-2*02, high levels of somatic hypermutation, the light chain 5-residue complementarity-determining region 3 (LCDR3), and the mimicry of Env-CD4 interactions. Other CD4b bNAbs have been characterized by their use of immunoglobulin heavy chain gene segment IGVH1-46 (e.g., IGVH1-46*01) and may have longer LCDR3s.

[0348] The present disclosure provides, among other things, polynucleotides encoding antibody agents, such as bNAbs, that target a broader group of HIV variants and can thus treat a greater number of HIV patients. In addition, the present disclosure provides compositions for the delivery of antibody agents, such as polynucleotides encoding bNAbs, that target various HIV sequences.

[0349] 1. Antiviral Therapy Against HIV HIV infection is currently primarily treated with antiretroviral therapy (ART). ART is a class of drugs that can reduce HIV proliferation, increase CD4 cell counts, and reduce the risk of transmission in infected individuals. The World Health Organization (WHO) recommends initiating ART in all HIV-infected adults, regardless of clinical stage or CD4 cell count (Consolidated guidelines on HIV prevention, testing, treatment, service delivery and monitoring: recommendations for a public health approach. Geneva: World Health Organization; 2021, incorporated herein by reference). However, ART is not a curative therapy, and viremia (e.g., viral load) quickly recurs when infected individuals stop taking ART. The high mutation rate of HIV also constrains patients in strictly adhering to treatment to avoid the emergence of escape mutants and treatment failure. Therefore, ART is intended to be taken daily for the entire life of the infected subject.

[0350] There are several classes of FDA-approved ARTs for treating HIV that act by different mechanisms. Effective management of HIV infection often requires a combination of at least three ARTs to treat the complex pathogenesis of the disease. The most effective combination of ARTs often varies between infected individuals (see, for example, Bhatti et al., Cureus 2016, which is incorporated herein by reference). Cihlar et al., Current opinion in virology, 2016, which is incorporated herein by reference in its entirety, reviews the classes of ART drugs for the treatment of HIV. [Table 1]

[0351] 2. HIV antibody drugs In addition to ART, anti-HIV antibodies have been developed. To use anti-HIV antibodies for treating HIV, generally, antibodies with specific characteristics are required, including safety, a favorable pharmacokinetic profile, very potent neutralizing activity, and broad neutralizing activity to effectively target the diversity present in HIV virions. Similar to other HIV therapeutics (e.g., including ART), viral escape from anti-HIV antibodies emerges as a significant challenge.

[0352] For example, there is Barouch, et al. who infected rhesus macaques with SHIV-SF162P3 (Barouch, et al., Nature 503:7475 224-228, 2013), which is incorporated herein by reference in its entirety. Here, the rhesus macaques were treated with three monoclonal antibodies (mAbs): PGT121, an N332 glycan-dependent mAb, 3BNC117, a CD4 binding site-specific mAb, and b12. The mAbs were administered on days 0 and 7 as a cocktail of 10 mg / kg each, or as a cocktail of 10 mg / kg each, or as a combination of only 10 mg / kg of PGT121 and 3BNC117 only on day 0. Transient viral suppression was observed until the level of bNAb dropped below 10 μg / mL. The mAbs were also administered to the monkeys alone. When PGT121 was administered alone, rapid virological suppression was achieved, but this rebounded in most animals after 6 - 8 weeks. Monkeys that received a combination of PGT121 and 3BNC117 were given a second dose on day 105, after the rebound of the viral level. Viral re-suppression was observed, but the suppression was not as persistent as the previous administration.

[0353] Shingai, et al. infected rhesus macaques with SHIV AD8EOdescribes being infected (Shingai, et al., Nature 503:7475 277-280, 2013, which is hereby incorporated by reference in its entirety). Here, rhesus monkeys were treated with 10-1074 and 3BNC117 mAbs alone or in combination. When 10 mg / kg was administered alone at 12 weeks post-inoculation, both antibodies resulted in rapid virus suppression, but the virus levels quickly rebounded. When both antibodies were combined and administered to chronically infected animals, the suppression period was longer and the CD4+ T cell levels improved, but the virus levels later rebounded. In other tests, both antibodies were found to pre-treat monkeys alone and prevent virus acquisition. Single-genome analysis of the virus that rebounded in monkeys treated with 10-074 revealed mutations that eliminate the N332 glycan of gp120, which exhibit resistance to the mAb. On the other hand, SGA analysis of the virus that rebounded in monkeys treated with both 10-074 and 3BNC117 revealed that not all monkeys contained viruses with changes that confer mAb resistance.

[0354] Caskey, et al. described the first-in-human dose-escalation phase 1 clinical trial of 3BNC117 (a CD4-binding site antibody) (Caskey, et al., Nature 522.7557:487-491, 2015, which is hereby incorporated by reference in its entirety). Uninfected and HIV-1-infected individuals were enrolled in the trial. 3BNC117 at doses of 1, 3, 10, or 30 mg / kg was generally safe and well tolerated, and no grade 3, 4, or severe adverse events were observed. In HIV-1-infected individuals, the antibody clearance rate was found to be faster than that in uninfected control subjects. The treatment effect on viral load was dose-dependent, and the viral load decreased by up to 2.5 logs at 10 and 30 mg / kg doses. Viral resistance developed in some individuals regardless of the mAb dose, while it was not observed in other individuals. Viral cloning and sequencing were performed, and G459D was a mutation commonly observed in the 10 mg / kg group, and other groups showed a longer V5 loop (with descriptions of other mutations). Both mutations may change the sensitivity to anti-CD4b.

[0355] Caskey, et al. also evaluated 10-074, a very potent mAb targeting the V3 loop of the HIV-1 envelope spike (Caskey et al., Nature Medicine 23.2:185-191, 2017), which is hereby incorporated by reference in its entirety. An open-label phase 1 first-in-human clinical trial was conducted on 14 uninfected individuals and 19 HIV-1-infected individuals. A single intravenous injection was administered at 3, 10, or 30 mg / kg. The mAb was generally safe and well tolerated, and no grade 3, 4, or severe adverse events were observed. In HIV-1-infected individuals, the antibody clearance rate was found to be faster than that in uninfected controls. Treatment suppressed the viral load in individuals with 10-074-sensitive strains, and then it rebounded. Single-genome sequencing (SGS) of the rebounded virus showed that all patients who responded to the treatment had PNGS at position N332 and no change in324 G(D / N)IR 327 was found to exhibit a motif. Four weeks after injection, 91% of the envelope sequences contained amino acid mutations, 97% of which eliminated the PNGS at position 332 by mutating either N332 or S334. 3% of the mutated sequences 324 G(D / N)IR 327 showed a change at D / N325 in the motif. Most of the mutations at the nucleic acid level were translocations, consistent with reverse transcriptase errors. Neutralization assay tests showed that mutant HIV-1 resistant to 10-074 was not resistant to 3BNC117, VRC01, or PGDM1400 (mAbs targeting other regions of HIV-1). SGS performed one week after injection revealed that resistant variants were already present or were rapidly generated.

[0356] Bar, et al. conducted two open-label trials regarding the safety, side effect profile, pharmacokinetic properties, and antiviral activity of VRC01 (a bNAb targeting the CD4 binding site of HIV) in patients who had undergone interruption of antiretroviral therapy (ART) (Bar et al., New England Journal of Medicine 375.21:2037-2050, 2016, which is hereby incorporated by reference in its entirety). In one trial, 40 mg / kg was injected three times over six weeks, and in the other trial, 40 mg / kg was injected eight times over six months. The treatment was well tolerated, and no grade 3 or higher adverse events were observed. Although sustained suppression of plasma viremia did not occur in either trial, a slight increase in the time to rebound was observed compared to past controls. Regardless of the time to rebound, resistance to VRC01 increased in almost all participants in one trial. Viral isolates showed higher resistance to VRC01 neutralization in pre-treatment samples compared to post-treatment samples. Treatment with VRC01 did not affect susceptibility to neutralization by other bNAbs.

[0357] Mendoza, et al. conducted a Phase 1b clinical trial to evaluate the combination of 3BNC117 and 10-1074, which were injected at weeks 0, 3, and 6 at a dose of 30 mg / kg (Mendoza, et al., Nature 561.7724:479-484, 2018, which is hereby incorporated by reference in its entirety). These two bNAbs target independent sites on the HIV-1 envelope spike. The injections were generally safe and well tolerated without reported serious adverse events. The median time to rebound was significantly prolonged by combination bNAb therapy. Two individuals with the earliest rebounds were found to have pre-existing resistant strains to one or the other bNAb. The rebounding virus was concentrated within a less diverse lineage, consistent with the outgrowth (escape) of one or two recrudescent viruses. Most of the rebounding viruses were found to contain mutations in 10-1074 as opposed to mutations in 3BNC117. Nevertheless, combination bNAb therapy was demonstrated to be more effective than single bNAb therapy in suppressing viral escape.

[0358] Gautam, et al. reported on SHIV AD8EORhesus monkeys were evaluated for infection by [[reference]] and treatment with 3BNC117-LS and 10-074-LS mAbs (Gautam, Rajeev, et al., Nature Medicine 24.5:610-616, 2018, which is hereby incorporated by reference in its entirety). M428L and N343S (collectively called LS) are mutations in the fragment domain of the mAb that extend the half-life. The LS mutations had no effect on virus neutralization in in vitro assays. The LS mAbs were administered alone at 20 mg / kg and were well tolerated in all monkeys. 10-1074-LS recipients showed enhanced protection against virus challenge compared to 3BNC117-LS recipients, but the LS mutations were more effective than the wild type in both antibodies. 10-1074-LS decayed more slowly than 3BNC117-LS in serum. It was determined that the concentration / neutralizing activity of the mAb predicted the probability of infection. Only experiments with virus challenge after pretreatment with the antibody were performed.

[0359] Shommers, et al. characterized an anti-HIV antibody called "1-18" in in vitro assays and in HIV-1-infected humanized mice. 1-18 was reported to bind to the CD4 binding site of HIV and have strong potency and broad neutralization against HIV strains. Schommers reported that 1-18 has certain features previously found in other anti-HIV antibodies, which may contribute to its potency and broad neutralization: (1) 1-18 has an aromatic residue that mimics the CD4 residue Phe43, which targets the "Phe43gp120 pocket", a feature previously reported for the anti-HIV antibody N6; (2) 1-18 contacts the adjacent gp120 promoter, as previously observed for the anti-HIV antibody 3BNC117, but with an increased buried surface area (due to the insertion of its 6 residues in CDRH1); and (3) a larger buried surface area on gp120 compared to other anti-HIV antibodies. In addition, 1-18 was reported to contact conserved residues on HIV gp120 that other anti-HIV antibodies did not contact. Schommers hypothesized that these contacts could make 1-18 less reliant on classical CD4 binding site contacts, thereby making viral escape more difficult. Nevertheless, Schommers recognized that a small number of HIV strains are resistant to 1-18.

[0360] In summary, the above data suggest that antibody administration may be effective in the treatment or prevention of HIV. However, the difficulty in targeting such mutant viruses is demonstrated by the above studies, as well as (1) the development of HIV resistance to therapy within weeks of using a single broad neutralizing antibody (bNAb) in therapy (Bar et al., Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption, N. Engl. J. Med. 375, 2037 - 2050 (2016), Caskey et al., Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature 522, 487 - 491 (2015), Caskey et al., Antibody 10-1074 suppresses viremia in HIV-1-infected individuals. Nat. Med. 23, 185 - 191 (2017), Klein et al., HIV therapy by a combination of broadly neutralizing antibodies in humanized mice, Nature 492, 118 - 122 (2012), Lynch et al., Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection, Sci. Transl. Med. 7, 319ra206 (2015), Scheid et al., HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption, Nature 535, 556 - 560 (2016), each of which is incorporated herein by reference in its entirety), and (2) the fact that certain combinations of antibodies have led to improved viral suppression by preventing the early development of resistance (Bar-On et al.,Safety and antiviral activity of combination HIV-1 broadly neutralizing antibodies in viremic individuals,Nat.Med.24,1701-1707(2018)、Klein et al.,2012、Mendoza et al.,Combination therapy with anti-HIV-1 antibodies maintains viral suppression,Nature 561,479-484(2018)、each of which is incorporated herein by reference in its entirety). It is clear from studies showing that viral rebounds observed for some of these antibodies may suggest that the antibodies are only effective for a limited period, for example, before HIV escape mutations develop.

[0361] Therefore, there is still a need for therapeutic and prophylactic therapies that can avoid viral escape and remain effective in HIV neutralization. As described herein, the present disclosure provides techniques useful in administering one or more antibody agents, such as polynucleotides encoding anti-HIV antibody agents. By using the techniques and methods described herein, it is possible, for example, to simultaneously produce different antibody agents from a polynucleotide. The format of the antibody agent is designed to minimize or eliminate the risk of mispairing of immunoglobulin chains. The ability to combine multiple antibody agent formulations (e.g., including 1-18 antibody agents) described herein allows for the development of compositions (e.g., pharmaceutical compositions) that deliver multiple antibody agents together such that they can bind to different epitopes of the HIV virus, thereby minimizing viral escape by mutation and improving overall effectiveness.

[0362] II. Polynucleotides for Delivery of Antibody Agents The present disclosure utilizes RNA technology, among other things, as a mode of direct expression for antibody agents as a novel class of antibody-based therapeutics. In some embodiments, the polynucleotides described herein encode the immunoglobulin chains of the antibody agent.

[0363] In some embodiments, the antibody agent targets HIV. In some embodiments, the antibody agent that targets HIV specifically binds to a specific epitope of an HIV polypeptide. For example, in some embodiments, the antibody agent specifically binds to an epitope that includes the CD4 binding site or a portion thereof. Reference is made to FIG. 2 by McCoy and Burton, Immunol Rev. 275.1 11-20, 2017, which is hereby incorporated herein by reference.

[0364] In some embodiments, the antibody agent has at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9It may have a binding affinity of M or less (e.g., measured by the dissociation constant). In some embodiments, the HIV antibody agent selectively binds to the target epitope of HIV, such that the binding between the HIV antibody agent and the target epitope is more than 2-fold, more than 5-fold, more than 10-fold, or more than 100-fold compared to the binding of the HIV antibody agent to the non-target epitope. In some embodiments, the HIV antibody agent may have a binding affinity for the HIV epitope and also for variants of the HIV epitope. One of ordinary skill in the art will fully understand that in some cases the binding affinity (e.g., measured by the dissociation constant) may be affected by non-covalent intermolecular interactions between two molecules, such as hydrogen bonding, electrostatic interactions, hydrophobicity, and van der Waals forces. Alternatively, or additionally, the binding affinity between a ligand and its target molecule may be affected by the presence of other molecules. One of ordinary skill in the art will be familiar with various techniques for measuring binding affinity and / or dissociation constant in accordance with the present disclosure, such as ELISA, gel shift assay, pull-down assay, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, diffraction grating binding interferometry, and spectroscopic assays.

[0365] In some embodiments, the anti-HIV antibody agent may comprise or be derived from a broadly neutralizing antibody (bNAb). In some embodiments, the anti-HIV antibody agent may be any one of the HIV-directed antibodies described in Barouch, et al., Nature 503:7475 224-228,2013, Shingai, et al., Nature 503:7475 277-280,2013, Caskey, et al., Nature 522.7557:487-491,2015, Caskey et al., Nature Medicine 23.2:185-191,2017, Bar et al., New England Journal of Medicine 375.21:2037-2050,2016, Mendoza, et al., Nature 561.7724:479-484,2018, Gautam, Rajeev, et al., Nature Medicine 24.5:610-616,2018, the entire contents of each of which are hereby incorporated by reference for the purposes described herein.

[0366] In some embodiments, the anti-HIV antibody agent can be, for example, 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, fragments thereof, or combinations thereof. Exemplary anti-HIV antibodies that can be used in the compositions described herein include, but are not limited to, 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, fragments thereof, or combinations thereof. For example, in some embodiments, the polynucleotide described herein can include one or more heavy chain complementarity determining regions (HCDRs) (e.g., HCDR1, HCDR2, and / or HCDR3) from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide described herein can include HCDR1, HCDR2, and HCDR3 from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide described herein can include the heavy chain variable domain from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. For example, in some embodiments, the polynucleotide described herein can include one or more light chain complementarity determining regions (LCDRs) (e.g., LCDR1, LCDR2, and / or LCDR3) from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide described herein can include LCDR1, LCDR2, and LCDR3 from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400.In some embodiments, the polynucleotides described herein can include light chain variable domains from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400.

[0367] In some embodiments, multiple polynucleotides, each encoding an immunoglobulin chain of an antibody agent, can be used to deliver two or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, or fragments or variants thereof) (e.g., by administration to a subject). In some embodiments, multiple polynucleotides, each encoding an immunoglobulin chain of an antibody agent, can be used to deliver three or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, or fragments or variants thereof) (e.g., by administration to a subject). In some embodiments, multiple polynucleotides, each encoding an immunoglobulin chain of an antibody agent, can be used to deliver four or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, or fragments or variants thereof) (e.g., by administration to a subject). In some embodiments, multiple polynucleotides, each encoding an immunoglobulin chain of an antibody agent, can be used to deliver two, three, four, five, or six antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, or fragments or variants thereof) (e.g., by administration to a subject).

[0368] In some embodiments, the antibody agent encoded by one or more of the polynucleotides described herein comprises all or a portion of 1-18 antibodies. In some embodiments, the antibody agent encoded by one or more of the polynucleotides provided herein comprises all or a portion of 1-18 antibodies. In some embodiments, the antibody agent comprises a heavy chain variable domain that includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof. In some embodiments, the antibody agent comprises a heavy chain variable domain that includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the antibody agent comprises a light chain variable domain that includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, the antibody agent comprises a light chain variable domain that includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, the antibody agent comprises (a) a heavy chain variable domain that includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and (b) a light chain variable domain that includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof.In some embodiments, the antibody agent comprises a heavy chain variable domain comprising (a) (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and a light chain variable domain comprising (b) (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).

[0369] In some embodiments, the polynucleotides described herein encode all or a portion of 1-18 antibodies. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain, and the heavy chain variable domain comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain, and the heavy chain variable domain comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6) and (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a light chain variable domain, and the light chain variable domain comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a light chain variable domain, and the light chain variable domain comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and the light chain variable domain comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and the light chain variable domain comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, the polynucleotides described herein encode two immunoglobulin chains, a first immunoglobulin chain comprising a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof, and a second immunoglobulin chain comprising a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof.In some embodiments, the polynucleotides described herein encode two immunoglobulin chains, a first immunoglobulin chain comprising a heavy chain variable domain that includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and a second immunoglobulin chain comprising a light chain variable domain that includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).

[0370] In some embodiments, the antibody agent encoded by one or more of the polynucleotides provided herein comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the antibody agent encoded by one or more of the polynucleotides provided herein comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the antibody agent comprises the heavy chain variable domain represented by SEQ ID NO: 24. In some embodiments, the antibody agent comprises the light chain variable domain represented by SEQ ID NO: 29.

[0371] In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 24, and the light chain variable domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 29.

[0372] In some embodiments, the polynucleotides described herein encode an immunoglobulin chain of an antibody agent, and the immunoglobulin chain comprises a heavy chain variable (VH) domain. In some embodiments, the VH domain comprises the VH domain of antibody 1-18. In some embodiments, the polynucleotide encodes the VH domain of an antibody selected from PGT121, 3BNC117, b12, 10-1074, 10-1074-LS, 10E8, VRC01, VRC07-523 or PGDM1400 (e.g., as described herein).

[0373] In some embodiments, the polynucleotide comprises a VH domain-encoding sequence comprising (a) an HCDR1-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 7, (b) an HCDR2-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 10, (c) an HCDR3-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 13, or (d) a combination thereof. In some embodiments, the polynucleotide comprises a VH domain-encoding sequence comprising (a) an HCDR1-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 7, (b) an HCDR2-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 10, and (c) an HCDR3-encoding sequence comprising the ribonucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide encodes a VH domain and comprises a VH-encoding sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25 or 27. In some embodiments, the polynucleotide encodes a VH domain and comprises a VH-encoding sequence according to SEQ ID NO: 25 or 27.

[0374] In some embodiments, the polynucleotides described herein encode an immunoglobulin chain of an antibody agent, and the immunoglobulin chain comprises a variable light (VL) domain. In some embodiments, the VL domain comprises the VL domain of antibody 1-18. In some embodiments, the polynucleotide encodes a VL domain of an antibody selected from PGT121, 3BNC117, b12, 10-1074, 10-1074-LS, 10E8, VRC01, VRC07-523 or PGDM1400 (e.g., as described herein).

[0375] In some embodiments, the polynucleotide comprises one or more coding regions encoding immunoglobulin chains of the antibody agent, and the immunoglobulin chain comprises a variable light (VL) domain. In some embodiments, the polynucleotide comprises a VL domain coding sequence comprising (a) an LCDR1 coding sequence comprising the ribonucleic acid sequence according to SEQ ID NO: 16, (b) an LCDR2 coding sequence comprising the ribonucleic acid sequence (GGCACCAGC) according to SEQ ID NO: 19, (c) an LCDR3 coding sequence comprising the ribonucleic acid sequence according to SEQ ID NO: 22, or (d) a combination thereof. In some embodiments, the polynucleotide comprises a VL domain coding sequence comprising (a) an LCDR1 coding sequence comprising the ribonucleic acid sequence according to SEQ ID NO: 16, (b) an LCDR2 coding sequence comprising the ribonucleic acid sequence (GGCACCAGC) according to SEQ ID NO: 19, and (c) an LCDR3 coding sequence comprising the ribonucleic acid sequence according to SEQ ID NO: 22. In some embodiments, the polynucleotide encodes a VL domain and comprises a VL coding sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 30. In some embodiments, the polynucleotide encodes a VL domain and comprises the VL coding sequence according to SEQ ID NO: 30.

[0376] In some embodiments, the antibody agent is formed by one, two, three or four immunoglobulin chains.

[0377] In some embodiments, the polynucleotides described herein encode a single immunoglobulin chain. In some embodiments, the first polynucleotide encodes the first immunoglobulin chain of the antibody agent. In some embodiments, the first polynucleotide encodes the first immunoglobulin chain of the antibody agent and the second polynucleotide encodes the second immunoglobulin chain of the antibody agent. In some embodiments, the first polynucleotide encodes the first immunoglobulin chain of the antibody agent, the second polynucleotide encodes the second immunoglobulin chain of the antibody agent, and the third polynucleotide encodes the third immunoglobulin chain of the antibody agent. In some embodiments, the first polynucleotide encodes the first immunoglobulin chain of the antibody agent, the second polynucleotide encodes the second immunoglobulin chain of the antibody agent, the third polynucleotide encodes the third immunoglobulin chain of the antibody agent, and the fourth polynucleotide encodes the fourth immunoglobulin chain of the antibody agent.

[0378] In some embodiments, the polynucleotides described herein encode two immunoglobulin chains. In some embodiments, a single polynucleotide can include a first coding region that encodes the first immunoglobulin chain of the antibody and a second coding region that encodes the second immunoglobulin chain of the antibody. In some embodiments, the first coding region and the second coding region are separated by an internal ribosome entry site (IRES), an internal promoter, or a peptide sequence, such as a "self-cleaving" 2A or 2A-like sequence (see, e.g., Szymczak et al. Nat Biotechnol 22:589, May 2004; ePub April 4 2004, which is incorporated herein by reference in its entirety) to produce the first immunoglobulin chain and the second immunoglobulin chain from the single polynucleotide.

[0379] The antibody agents encoded by one or more polynucleotides described herein can be in various forms described herein. Exemplary types of antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent can include one or more sequence elements that have been humanized, chimerized, etc., as known in the art. The antibody agents utilized in accordance with the present disclosure, in some embodiments, include, but are not limited to, unmodified IgG, IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybody®, etc.); CrossMab (e.g., CrossMab CH1-CLx , CrossMab CH1-CLcv , bispecific CrossMab with knob-in-hole CH1-CLx ); antibody fragments, e.g., Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated complementarity-determining regions (CDRs) or combinations thereof; single-chain Fv (scFv); scFv-Fc fusions; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, e.g., IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probody®), small modular immunopharmaceuticals (“SMIP™”); single-chain or tandem diabodies (TandAb®); VHH; anticalin®; nanobody® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; avimer®; DART; TCR-like antibodies; adnectin®; affilin®; transbody®; affibody®; TrimerX®; microprotein; finomer®; centyrin®; and forms selected from KALBITOR®. In some embodiments, the immunoglobulin chains and / or fragments of such antibodies can be used in combination, e.g., by combining an scFv-Fc arm with a conventional antibody arm.

[0380] Exemplary forms that can be used in accordance with the present disclosure are further described below.

[0381] A. Conventional Antibodies In some embodiments, the polynucleotides described herein can be used to express conventional antibodies. As used herein, "conventional antibody" refers to an antibody agent comprising two heavy chains and two light chains (see, e.g., FIGS. 5A and 4A). Each heavy chain comprises a heavy chain variable domain operably linked to one or more heavy chain constant domains. In some embodiments, the one or more heavy chain constant domains comprise a CH1 domain, a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Optionally, the one or more heavy chain constant domains comprise a CH1 domain, a hinge domain, a CH2 domain, a CH3 domain, a CH4 domain, or a combination thereof. Each light chain comprises a light chain variable domain operably linked to a light chain constant domain.

[0382] Typically, the heavy chain variable domain and the light chain variable domain can be further subdivided into regions of variability called complementarity determining regions (CDRs) that are separated by more conserved regions called framework regions (FRs). Such heavy chain variable domains and light chain variable domains can each comprise, for example, three CDRs and four framework regions arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus towards the carboxyl terminus, although one or more of these can be engineered as described herein. The CDRs in the heavy chain are called "HCDR1", "HCDR2", and "HCDR3", respectively, and the CDRs in the light chain are called "LCDR1", "LCDR2", and "LCDR3".

[0383] Conventional antibodies described herein may include any one of the five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In some embodiments, the conventional antibody includes an IgG or IgA antibody. In some embodiments, the conventional antibodies described herein include a particular isotype selected from the group of IgA and IgG isotypes: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Further, in some embodiments, the conventional antibody may include any particular heavy chain constant domain corresponding to a different class of immunoglobulin, each containing α, δ, ε, γ, and μ, respectively. In some embodiments, the conventional antibody is an unmodified IgG1 antibody or another antibody class or isotype as described herein. (See, e.g., Hudson et al., Nat. Med., 9:129-134 (2003), Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994), Hollinger et al. Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), WO93 / 01161, and U.S. Patent Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458, each of which is incorporated herein by reference). In addition to the various isotypes, the IgG subclass has allelic diversity that gives rise to allotype variants or allotypes. The IgG antibody agents described herein may include, but are not limited to, particular allotypes including G1m3, Glm17, G1m17,1, or G1m17,1,2, or G1m3,1 (see Vidarsson et al., Front. Immunol, 5(520):1-17, 2014, which is incorporated herein by reference in its entirety).

[0384] Conventional antibody Fc regions bind to elements of the complement system and also to receptors on effector cells, such as receptors on effector cells that mediate cytotoxicity. As is known in the art, the affinity of the Fc region for Fc receptors and / or other binding attributes can be modulated by glycosylation or other modifications. In some embodiments, conventional antibodies produced and / or utilized in accordance with the present invention include a glycosylated Fc domain, which includes, for example, an Fc domain having such glycosylation that has been modified or engineered. In some embodiments, conventional antibodies are produced naturally (e.g., produced by an organism that reacts to an antigen) or by recombinant manipulation, chemical synthesis, or other artificial systems or methodologies. In some embodiments, conventional antibodies are polyclonal, and in some embodiments, conventional antibodies are monoclonal. In some embodiments, conventional antibodies have a constant region sequence characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, conventional antibody sequence elements have been humanized, chimerized, etc., as is known in the art.

[0385] Conventional antibodies described herein are antibodies that have a structure substantially similar to the native antibody structure or have a heavy chain containing an Fc region as defined herein.

[0386] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides provided herein include all or a portion of the 1-18 antibodies. In some embodiments, the conventional antibody includes a heavy chain variable domain that comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof. In some embodiments, the conventional antibody includes a heavy chain variable domain that comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the conventional antibody includes a light chain variable domain that comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, the conventional antibody includes a light chain variable domain that comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, the conventional antibody includes (a) a heavy chain variable domain that comprises (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and (b) a light chain variable domain that comprises (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof.In some embodiments, the conventional antibody comprises a heavy chain variable domain comprising (a) (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and a light chain variable domain comprising (b) (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).

[0387] In some embodiments, the polynucleotides described herein encode the immunoglobulin chains of a conventional antibody, the immunoglobulin chain includes a heavy chain variable domain, and the heavy chain variable domain includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof. In some embodiments, the polynucleotides described herein encode the immunoglobulin chains of a conventional antibody, the immunoglobulin chain includes a heavy chain variable domain, and the heavy chain variable domain includes (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the polynucleotides described herein encode the immunoglobulin chains of a conventional antibody, the immunoglobulin chain includes a light chain variable domain, and the light chain variable domain includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, the polynucleotides described herein encode the immunoglobulin chains of a conventional antibody, the immunoglobulin chain includes a light chain variable domain, and the light chain variable domain includes (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).In some embodiments, the polynucleotide described herein encodes two immunoglobulin chains of a conventional antibody, a first immunoglobulin chain comprising a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and a second immunoglobulin chain comprising a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, the polynucleotide described herein encodes two immunoglobulin chains of a conventional antibody, a first immunoglobulin chain comprising a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and a second immunoglobulin chain comprising a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).

[0388] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides provided herein include a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides provided herein include a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the conventional antibody includes the heavy chain variable domain represented by SEQ ID NO: 24. In some embodiments, the conventional antibody includes the light chain variable domain represented by SEQ ID NO: 29.

[0389] In some embodiments, the polynucleotides described herein encode an immunoglobulin chain of a conventional antibody, and the immunoglobulin chain includes a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain of a conventional antibody, and the immunoglobulin chain includes a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 29.

[0390] Conventional antibodies encoded by one or more of the polynucleotides described herein may include one or more heavy chain constant domains. In some embodiments, the one or more heavy chain constant domains include a CH3 domain. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH3 domain that includes the G1m3, G1m17 or Glm17,1 allotype. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in any one of SEQ ID NOs: 68, 71, 74, 77, 80, 83, 86 or 89. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH3 domain having the amino acid sequence set forth in any one of SEQ ID NOs: 68, 71, 74, 77, 80, 83, 86 or 89.

[0391] Conventional antibodies encoded by one or more of the polynucleotides described herein may include one or more heavy chain constant domains that include amino acid modifications (e.g., substitutions or deletions) at one or more amino acid positions. For example, conventional antibodies encoded by one or more of the polynucleotides described herein may include an L / S mutation within the CH3 region (to enhance FcRn binding) (see Zalevsky J et al. Nat Biotechnol. 2010, which is incorporated herein by reference). Such mutations are designated M428L and N434S based on EU numbering and are referred to herein as "LS" or "L / S" (see, e.g., FIG. 5C). In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a deletion of E294 (for enhanced sialic acid addition to Fc) (see Bas M et al. J Immunol 2019, which is incorporated herein by reference).

[0392] The present disclosure also provides techniques that can be used to express antibody agents, such as those shown in FIG. 3, or as described in Stadler et al. (2016) Oncoimmunology 5(3):e1091555 and / or Stadler et al. (2017) Nature Medicine 23(7):815-817. There are difficulties in producing multiple antibody agents from a single composition (e.g., a composition containing a polynucleotide sufficient to encode multiple antibody agents). This is because, in particular, the random pairing of different antibody heavy and light chains can result in unwanted antibody species. Due to the presence of mispaired by-products and a significant decrease in production yield, sophisticated purification procedures are required to isolate the desired antibody agents under such circumstances (see, e.g., Morrison, S.L., Nature Biotech. 25, 1233-1234, 2007, which is incorporated herein by reference). Generally, when using recombinant expression techniques, the same problem of mispaired by-products remains. One approach to solving the problem of mispaired by-products is known as the "knob-into-hole technology" (KIH), which aims to force the pairing of two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. On one chain, bulky amino acids are replaced with amino acids having short side chains to create a "hole", and on the other CH3 domain, amino acids having large side chains are introduced to create a "knob". By co-expressing these two heavy chains with two light chains, the formation of heterodimers was observed at a higher yield compared to homodimers (see Ridgway, J.B., et al, Protein Eng. 9, 617-621, 1996, and WO96 / 027011, which are incorporated herein by reference). In some embodiments, the antibody agents described herein utilize the KIH technology, which is described, for example, in WO1998 / 050431, the entire disclosure of which is incorporated herein by reference. As described herein, the antibody agents can include specific mutations that utilize the KIH technology, including but not limited to modifications of CH3.In some embodiments, the antibody agent comprises a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). In some embodiments, the antibody agent comprises a CH3 domain, and the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). Such a combination of mutations is referred to herein as "cah". In some embodiments, the antibody agent comprises a CH3 domain comprising one or more mutations selected from S354C and T366W (based on EU numbering). In some embodiments, the antibody agent comprises a CH3 domain comprising each of the following mutations: S354C and T366W (based on EU numbering). Such a combination of CH3 mutations is referred to herein as "cak".

[0393] Thus, in some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises a CH3 domain comprising one or more mutations selected from S354C and T366W (based on EU numbering).

[0394] In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: M428, N434S, or a combination thereof (e.g., the "L / S" mutation). In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence comprising any one of SEQ ID NOs: 75 and 78. In some embodiments, the polynucleotide encodes a CH3 domain comprising one or more of the following substitution mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NOs: 81 and 84. In some embodiments, the polynucleotide encodes a CH3 domain comprising either or both of the following substitution mutations: S354C and T366W (based on EU numbering). In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NOs: 87 and 90.

[0395] In some embodiments, the polynucleotide encodes an immunoglobulin chain in which the VH domain is operably linked to one or more constant domains, and the one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO: 69. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence encoding a CH3 domain comprising the G1m3, G1m17 or Glm17,1 allotype. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence comprising any one of SEQ ID NOs: 69 and 72.

[0396] In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH1 domain. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH1 domain that includes the G1m3 allotype. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH1 domain that includes the G1m17 allotype. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein include a CH1 domain that includes the amino acids set forth in SEQ ID NO: 38 or 41.

[0397] In some embodiments, the polynucleotide encodes an immunoglobulin chain that includes a VH domain operably linked to one or more constant domains, and the one or more constant domains include a CH1 domain. In some embodiments, the polynucleotide includes the CH1 ribonucleic acid sequence according to SEQ ID NO: 39. In some embodiments, the polynucleotide encodes a CH1 domain that includes the G1m3 allotype. In some embodiments, the polynucleotide encodes a CH1 domain that includes the G1m17 allotype. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence according to SEQ ID NO: 39 or 42.

[0398] In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more mutations. In some embodiments, the polynucleotide encodes a CH1 domain that contains the addition of one or more serine residues. In some embodiments, the polynucleotide encodes a CH1 domain that contains the addition of two additional serine residues (referred to herein as "SS"). In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45 or 48. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence represented by SEQ ID NO: 45 or 48. In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more charge variant mutations. In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more substitution mutations selected from K147E, K213D or combinations thereof. In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 51 or 866. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence by SEQ ID NO: 51 or 866.

[0399] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein contain a hinge domain. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein contain a hinge domain (referred to herein as "hinge" in Tables 2 and 4) that contains the amino acid sequence represented by SEQ ID NO: 104.

[0400] In some embodiments, the polynucleotide encodes a hinge domain. In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 105. In some embodiments, the polynucleotide encodes a hinge domain comprising an amino acid modification comprising a deletion of one or more amino acid residues. In some embodiments, the polynucleotide encodes a hinge domain comprising an amino acid modification comprising a deletion of the amino acid residues EPKSC in the conventional Ig hinge domain (represented by SEQ ID NO: 104). Such a modification is referred to herein as "hinge_del" or "ΔEPKSC". In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 111. In some embodiments, the polynucleotide encodes a hinge domain comprising an amino acid modification comprising the C220S mutation (based on EU numbering). Such a mutant hinge domain is referred to herein as "hinge_S" or "C / S". In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 108.

[0401] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein have a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that represented by SEQ ID NO: 53. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain having the amino acid sequence represented by SEQ ID NO: 53.

[0402] In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises a CH2 domain having one or more mutations (e.g., with respect to SEQ ID NO: 53). For example, in some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises one or more of the following mutations: G236A, A330L, and I332E (based on EU numbering). For example, in some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises the following mutations, referred to herein as "GAALIE": G236A, A330L, and I332E (based on EU numbering). Such mutations in the CH2 domain have been associated with increased affinity for the Fc receptors FcgRIIA and FcgRIII to enhance the effector function of the antibody.

[0403] In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 56. In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises a CH2 domain having the amino acid sequence set forth in SEQ ID NO: 56.

[0404] In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise one or more mutations selected from G236A and I332E (based on EU numbering). In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise mutations selected from G236A and I332E (based on EU numbering), referred to herein as "GAIE". In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 59. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain having the amino acid sequence set forth in SEQ ID NO: 59.

[0405] In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise the mutation G236A (based on EU numbering), referred to herein as "GA". In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 62. In some embodiments, conventional antibodies encoded by one or more of the polynucleotides described herein comprise a CH2 domain having the amino acid sequence set forth in SEQ ID NO: 62.

[0406] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein include the variant I332E (based on EU numbering), referred to herein as "IE". In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein include a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein include a CH2 domain having the amino acid sequence set forth in SEQ ID NO: 65.

[0407] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operably linked to one or more constant domains, and the one or more constant domains include a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO: 54. In some embodiments, the CH2 ribonucleic acid encodes a CH2 domain having one or more amino acid substitution mutations. For example, in some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations herein referred to as "GAALIE": G236A, A330L, and I332E (based on EU numbering). Such mutations in the CH2 domain have been associated with increased affinity for the Fc receptors FcgRIIA and FcgRIII to enhance the effector function of the antibody. In some embodiments, the CH2 ribonucleic acid sequence comprises or consists of the sequence according to SEQ ID NO: 57. In some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations herein referred to as "GAIE": G236A and I332E (based on EU numbering). In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 60. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation G236A (based on EU numbering) herein referred to as "GA". In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 63. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation I332E (based on EU numbering) herein referred to as "IE". In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 66. In some embodiments, the CH2 ribonucleic acid sequence encodes a CH2 domain comprising a deletion of E294 (based on EU numbering).

[0408] In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein comprise a signal peptide that includes a human signal peptide. In some embodiments, the conventional antibodies encoded by one or more of the polynucleotides described herein comprise a signal peptide that includes SEQ ID NO: 1.

[0409] In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes a light chain constant domain, and the light chain constant domain includes a kappa light chain constant domain. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein has a kappa light chain constant domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes a kappa light chain constant domain having the amino acid sequence represented by SEQ ID NO: 92. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes a lambda chain variable domain.

[0410] In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes an immunoglobulin chain (e.g., an immunoglobulin heavy chain) encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences represented by SEQ ID NOs: 113 - 160. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes an immunoglobulin chain (e.g., an immunoglobulin heavy chain) encoded by a nucleic acid sequence represented by any one of SEQ ID NOs: 113 - 160. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes an immunoglobulin chain (e.g., an immunoglobulin light chain) encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 449. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein includes an immunoglobulin chain (e.g., an immunoglobulin light chain) encoded by the nucleic acid sequence represented by SEQ ID NO: 449.

[0411] In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence represented by any one of SEQ ID NOs: 613, 616, 622 and 625. In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence represented by SEQ ID NO: 619. In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) encoded by a nucleic acid sequence represented by any one of SEQ ID NOs: 613, 616, 622 and 625. In some embodiments, a conventional antibody encoded by one or more of the polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) encoded by a nucleic acid sequence represented by SEQ ID NO: 619.

[0412] In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence represented by any one of SEQ ID NOs: 614, 617, 623 and 626. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) comprising an amino acid sequence represented by any one of SEQ ID NOs: 614, 617, 623 and 626. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 620. In some embodiments, a conventional antibody encoded by one or more polynucleotides described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) comprising the amino acid sequence represented by SEQ ID NO: 620.

[0413] The composition of exemplary immunoglobulin chains (e.g., immunoglobulin heavy or light chains) of conventional antibodies described herein is shown in Table 2 below.

Table 2-1

Table 2-2

Table 2-3

[0414] B.CrossMab CH1-CLx The present disclosure also provides techniques that can be used to deliver and express the antibody agents described herein in the "CrossMab" format (see, e.g., WO2015 / 101588A1, WO2009 / 080253A1, and Schaefer, W. et al, PNAS, 108, 11187-1191, 2011, which are hereby incorporated by reference in their entirety). In some embodiments, the antibody agent in the CrossMab format contains a CL-CH1 crossover in one or both binding arms (referred to herein as "CrossMab" CH1-CLx " or "CH1-CLx"). Such modifications reduce the formation of by-products resulting from the mispairing of the light chain of the first antibody that specifically binds to the first antigen and the heavy chain of the second antibody that specifically binds to the wrong second antigen (compared to techniques without such domain exchange).

[0415] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein comprises a first immunoglobulin chain and a second immunoglobulin chain. In some embodiments, the polynucleotide can encode the first and second immunoglobulin chains of the CrossMab CH1-CLx antibody agent described herein. In some embodiments, the polynucleotide encoding the first immunoglobulin chain comprises ribonucleic acid sequences encoding a VH domain, a CL domain, a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the polynucleotide encoding the second immunoglobulin chain comprises ribonucleic acid sequences encoding a variable light (VL) domain and a CH1 domain (see, e.g., Figure 4B). In some embodiments, the polynucleotide encoding the CrossMab CH1-CLx antibody agent comprises ribonucleic acid sequences encoding any one of the immunoglobulin chain configurations in Table 3 corresponding to SEQ ID NOs: 161-208. In some embodiments, the polynucleotide encoding the CrossMab CH1-CLx drug antibody comprises ribonucleic acid sequences encoding any one of the immunoglobulin chain configurations in Table 3 corresponding to SEQ ID NOs: 450 and 451.

[0416] In some embodiments, CrossMab CH1-CLx The antibody agent may be encoded by two separate polynucleotides, a first polynucleotide (see, e.g., FIG. 9A) comprising a coding region encoding, in order from 5' to 3', a heavy chain variable domain (VH), a light chain constant region (CL), a hinge region, a CH2 domain, and a CH3 domain, and a second polynucleotide (see, e.g., FIG. 9B) comprising a coding region encoding, in order from 5' to 3', a light chain variable domain (VL) and a CH1 domain.

[0417] In some embodiments, the CrossMab encoded by one or more polynucleotides provided herein CH1-CLx The antibody agent includes all or a portion of the 1-18 antibodies. In some embodiments, CrossMab CH1-CLx The antibody agent includes a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof. In some embodiments, CrossMab CH1-CLx The antibody agent includes a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, CrossMab CH1-CLx The antibody agent includes a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, CrossMab CH1-CLx The antibody agent includes a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, CrossMabCH1-CLx The antibody agent comprises: (a) a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and (b) a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), or (iv) a combination thereof. In some embodiments, CrossMab CH1-CLx The antibody agent comprises: (a) a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12); and (b) a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21).

[0418] In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx encoding an immunoglobulin chain of the antibody agent, the immunoglobulin chain comprising a heavy chain variable domain, the heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof. In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx encoding an immunoglobulin chain of the antibody agent, the immunoglobulin chain comprising a heavy chain variable domain, the heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the polynucleotide described herein is CrossMabCH1-CLx Encoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising a light chain variable domain, the light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21) or (iv) a combination thereof. In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx Encoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising a light chain variable domain, the light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18) and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, the polynucleotide described herein is a first immunoglobulin chain comprising a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12) or (iv) a combination thereof, and a second immunoglobulin chain comprising a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21) or (iv) a combination thereof, the two CrossMab CH1-CLxEncodes the immunoglobulin chains of the antibody agent. In some embodiments, the polynucleotide described herein is a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), and a first immunoglobulin chain comprising the heavy chain variable domain; and a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21), and a second immunoglobulin chain comprising the light chain variable domain, two CrossMabs CH1-CLx Encodes the immunoglobulin chains of the antibody agent.

[0419] In some embodiments, the CrossMab encoded by one or more polynucleotides provided herein CH1-CLx The antibody agent comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the CrossMab encoded by one or more polynucleotides provided herein CH1-CLx The antibody agent comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the CrossMab encoded by one or more polynucleotides provided herein CH1-CLx The antibody agent comprises the heavy chain variable domain represented by SEQ ID NO: 24. In some embodiments, the CrossMab encoded by one or more polynucleotides provided herein CH1-CLx The antibody agent comprises the light chain variable domain represented by SEQ ID NO: 29.

[0420] In some embodiments, the polynucleotide described herein is a CrossMab CH1-CLxEncoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 24. In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx Encoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence represented by SEQ ID NO: 29. In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx Encoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising the heavy chain variable domain represented by SEQ ID NO: 24. In some embodiments, the polynucleotide described herein is CrossMab CH1-CLx Encoding an immunoglobulin chain of an antibody agent, the immunoglobulin chain comprising the light chain variable domain represented by SEQ ID NO: 29.

[0421] As described above, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent may comprise one or more heavy chain constant domains. In some embodiments, the one or more heavy chain constant domains comprise a CH3 domain. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises a CH3 domain comprising the G1m3, G1m17 or Glm17,1 allotype. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLxThe antibody agent comprises a CH3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence represented by any one of SEQ ID NOs: 68, 71, 74, 77, 80, 83, 86 or 89. In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises a CH3 domain having an amino acid sequence represented by any one of SEQ ID NOs: 68, 71, 74, 77, 80, 83, 86 or 89.

[0422] CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent may comprise one or more heavy chain constant domains containing amino acid modifications (e.g., substitutions or deletions) at one or more amino acid positions. For example, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent may contain an L / S mutation within the CH3 region (to enhance FcRn binding; see Zalevsky J et al. Nat Biotechnol. 2010, which is incorporated herein by reference). Such mutations are designated M428L and N434S based on EU numbering and are referred to herein as "LS" or "L / S" (see, e.g., FIG. 5C). In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent contains a deletion of E294 (for enhanced sialic acid addition to Fc; see Bas M et al. J Immunol 2019, which is incorporated herein by reference).

[0423] In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLxThe antibody agent comprises a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). This combination of mutations is referred to herein as "cah". In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises a CH3 domain comprising one or more mutations selected from S354C and T366W (based on EU numbering). This combination of CH3 mutations is referred to herein as "cak".

[0424] In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: M428, N434S, or a combination thereof (e.g., the "L / S" mutation). In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence comprising any one of SEQ ID NOs: 75 and 78. In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: Y349C, T366S, L368A, and Y407V (based on EU numbering). In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NOs: 81 and 84. In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: S354C and T366W (based on EU numbering). In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NOs: 87 and 90.

[0425] In some embodiments, the polynucleotide encodes an immunoglobulin chain in which the VH domain is operably linked to one or more constant domains, and the one or more constant domains include a CH3 domain. In some embodiments, the polynucleotide includes a ribonucleic acid sequence according to SEQ ID NO: 69. In some embodiments, the polynucleotide includes a CH3 ribonucleic acid sequence encoding a CH3 domain that includes the G1m3, G1m17 or Glm17,1 allotype. In some embodiments, the polynucleotide includes a CH3 ribonucleic acid sequence that includes any one of SEQ ID NOs: 69 and 72.

[0426] In some embodiments, the CrossMab CH1-CLx antibody agent encoded by one or more of the polynucleotides described herein includes a light chain constant domain. In some embodiments, the light chain constant domain includes a kappa light chain constant domain. In some embodiments, the CrossMab CH1-CLx antibody agent encoded by one or more of the polynucleotides described herein includes a kappa light chain constant domain having an amino acid sequence that is at least 80, 85, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92. In some embodiments, the CrossMab CH1-CLx antibody agent encoded by one or more of the polynucleotides described herein includes a kappa light chain constant domain having the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the CrossMab CH1-CLx antibody agent includes a lambda chain variable domain.

[0427] In some embodiments, the CrossMab CH1-CLx antibody agent encoded by one or more of the polynucleotides described herein includes a hinge domain. In some embodiments, the CrossMab CH1-CLxThe antibody agent contains a hinge domain (referred to as "hinge" in Tables 2 and 4 herein) containing the amino acid sequence represented by SEQ ID NO: 104.

[0428] In some embodiments, the polynucleotide encodes a hinge domain. In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 105. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification that includes a deletion of one or more amino acid residues. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification that includes a deletion of the amino acid residues EPKSC in the conventional Ig hinge domain (represented by SEQ ID NO: 104). Such a modification is referred to herein as "hinge_del" or "ΔEPKSC". In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 111. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification that includes a C220S mutation (based on EU numbering). Such a mutant hinge domain is referred to herein as "hinge_S" or "C / S". In some embodiments, the polynucleotide encodes a hinge ribonucleic acid sequence represented by SEQ ID NO: 108.

[0429] In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent contains a CH2 domain. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that represented by SEQ ID NO: 53. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent contains a CH2 domain having the amino acid sequence represented by SEQ ID NO: 53.

[0430] In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx antibody agent comprises a CH2 domain having one or more mutations (e.g., with respect to SEQ ID NO: 53). For example, in some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx antibody agent comprises one or more of the following mutations: G236A, A330L, and I332E (based on EU numbering). Such combinations of mutations are referred to herein as "GAALIE". Such mutations in the CH2 domain have been associated with an increased affinity for the Fc receptors FcgRIIA and FcgRIII to enhance the effector function of the antibody.

[0431] In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx antibody agent comprises a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 56. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx antibody agent comprises a CH2 domain having the amino acid sequence set forth in SEQ ID NO: 56.

[0432] In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx antibody agent comprises one or more mutations selected from G236A and I332E (based on EU numbering). Such combinations of CH2 mutations are referred to herein as "GAIE". In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLxThe antibody agent contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 59. In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains a CH2 domain having the amino acid sequence represented by SEQ ID NO: 59.

[0433] In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains the mutation G236A (based on EU numbering) herein referred to as "GA". In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 62. In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains a CH2 domain having the amino acid sequence represented by SEQ ID NO: 62.

[0434] In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains the mutation I332E (based on EU numbering) herein referred to as "IE". In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65. In some embodiments, it is a CrossMab encoded by one or more of the polynucleotides described herein. CH1-CLx The antibody agent contains a CH2 domain having the amino acid sequence represented by SEQ ID NO: 65.

[0435] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operably linked to one or more constant domains, and the one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO: 54. In some embodiments, the CH2 ribonucleic acid encodes a CH2 domain having one or more amino acid substitution mutations. For example, in some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations herein referred to as "GAALIE": G236A, A330L, and I332E (based on EU numbering). Such mutations in the CH2 domain have been associated with an increased affinity for the Fc receptors FcgRIIA and FcgRIII for enhancing the effector function of the antibody. In some embodiments, the CH2 ribonucleic acid sequence comprises or consists of the sequence according to SEQ ID NO: 57. In some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations herein referred to as "GAIE": G236A and I332E (based on EU numbering). In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 60. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation G236A (based on EU numbering) herein referred to as "GA". In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 63. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation I332E (based on EU numbering) herein referred to as "IE". In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO: 66. In some embodiments, the CH2 ribonucleic acid sequence encodes a CH2 domain comprising an E294 deletion (based on EU numbering) herein referred to as "E294del".

[0436] In some embodiments, the CrossMab CH1-CLx antibody agent encoded by one or more of the polynucleotides described herein comprises a CH1 domain. In some embodiments, the CrossMabCH1-CLx The antibody agent includes a CH1 domain containing the G1m3 allotype. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent includes a CH1 domain containing the G1m17 allotype. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent includes a CH1 domain containing the amino acids represented by SEQ ID NO: 38 or 41.

[0437] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operably linked to one or more constant domains, and the one or more constant domains include a CH1 domain. In some embodiments, the polynucleotide includes the CH1 ribonucleic acid sequence according to SEQ ID NO: 39. In some embodiments, the polynucleotide encodes a CH1 domain containing the G1m3 allotype. In some embodiments, the polynucleotide encodes a CH1 domain containing the G1m17 allotype. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence according to SEQ ID NO: 39 or 42.

[0438] In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more mutations. In some embodiments, the polynucleotide encodes a CH1 domain that contains the addition of one or more serine residues. In some embodiments, the polynucleotide encodes a CH1 domain that contains the addition of two additional serine residues (referred to herein as "SS"). In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45 or 48. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence represented by SEQ ID NO: 45 or 4. In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more charge variant mutations. In some embodiments, the polynucleotide encodes a CH1 domain that contains one or more substitution mutations selected from K147E, K213D or combinations thereof. In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 51 or 866. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence by SEQ ID NO: 51 or 866.

[0439] In some embodiments, the CrossMab CH1-CLx encoded by one or more of the polynucleotides described herein includes a signal peptide that includes a husec2 signal peptide. In some embodiments, the CrossMab CH1-CLx encoded by one or more of the polynucleotides described herein includes a signal peptide that includes SEQ ID NO: 1.

[0440] In some embodiments, the CrossMab CH1-CLxThe antibody agent comprises an immunoglobulin chain encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences represented by SEQ ID NOs: 161 to 208 (heavy chain) and SEQ ID NOs: 450 and 451 (light chain). In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises an immunoglobulin chain encoded by a nucleic acid sequence represented by any one of SEQ ID NOs: 161 to 208 (heavy chain) and SEQ ID NOs: 450 and 451 (light chain).

[0441] In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises an immunoglobulin chain encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences represented by SEQ ID NO: 628 (heavy chain) and / or SEQ ID NO: 631 (light chain). In some embodiments, CrossMab CH1-CLx comprises an immunoglobulin chain represented by SEQ ID NO: 628 (heavy chain) and / or SEQ ID NO: 631 (light chain).

[0442] In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises an immunoglobulin chain (e.g., immunoglobulin heavy chain) comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence represented by SEQ ID NO: 629. In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises an immunoglobulin chain (e.g., immunoglobulin heavy chain) comprising an amino acid sequence represented by any one of SEQ ID NO: 629. In some embodiments, CrossMab encoded by one or more of the polynucleotides described hereinCH1-CLx The antibody agent comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 632. In some embodiments, the CrossMab encoded by one or more of the polynucleotides described herein CH1-CLx The antibody agent comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) having the amino acid sequence represented by SEQ ID NO: 632. The CrossMab described herein CH1-CLx Exemplary heavy and light chain compositions of the antibody agent are shown in Table 3 below.

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

[0443] C. CrossMab CH1-CLcv In some embodiments, the antibody agent described herein is in a form in which one or more charge variants (cv) are introduced into domains (e.g., constant domains, e.g., CH1 domain, CL domain or combinations thereof). Such a form is referred to herein as "CrossMab" CH1-CLcvIt is also referred to as "」 or "CH1-cv". In some embodiments, such an antibody agent contains charge variants in both arms of the antibody (see, for example, FIG. 4C). Exemplary charge variants are described, for example, in WO2017055539A1, which is hereby incorporated by reference in its entirety. In some embodiments, in contrast to the form described in WO2017055539A1, the described CH1-cv antibody agent does not contain the exchange of the CH1 domain and the CL domain of the antibody in either arm.

[0444] In some embodiments, a charge variant (cv) is introduced into the CH1 and / or CL domain of the antibody agent to prevent mispairing of the immunoglobulin chains of the antibody agent. Such charge variants can include, for example, introducing one or more positively charged amino acid residues into the CH1 domain and one or more negatively charged amino acid residues into the CL at specific positions at the interface between CH1 and CL, or vice versa.

[0445] In some embodiments, the polynucleotide can encode the heavy chain and / or light chain of the antibody agent described herein. CH1-CLcv The antibody agent may be encoded by two separate polynucleotides: a first polynucleotide (see, for example, FIG. 10A) containing a coding region that encodes, in order from 5' to 3', a heavy chain variable domain (VH), a CH1 domain containing one or more charge variants described herein, a hinge region, a CH2 domain, and a CH3 domain; and a second polynucleotide (see, for example, FIG. 10B) containing a coding region that encodes, in order from 5' to 3', a light chain variable domain (VL) and a light chain constant domain (CL) containing one or more charge variants described herein.

[0446] In some embodiments, the CrossMab CH1-CLcv The antibody agent may be encoded by two separate polynucleotides: a first polynucleotide (see, for example, FIG. 10A) containing a coding region that encodes, in order from 5' to 3', a heavy chain variable domain (VH), a CH1 domain containing one or more charge variants described herein, a hinge region, a CH2 domain, and a CH3 domain; and a second polynucleotide (see, for example, FIG. 10B) containing a coding region that encodes, in order from 5' to 3', a light chain variable domain (VL) and a light chain constant domain (CL) containing one or more charge variants described herein.

[0447] In some embodiments, the CrossMab CH1-CLcvThe antibody agent comprises a first immunoglobulin chain, the first immunoglobulin chain comprises a CL domain, and the CL domain comprises an amino acid at position 123 (EU numbering) substituted with an amino acid selected from K, R, and H. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a first immunoglobulin chain, the first immunoglobulin chain comprises a CL domain, and the CL domain comprises an amino acid at position 124 (EU numbering) substituted with an amino acid selected from K, R, and H. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a second immunoglobulin chain, and the second immunoglobulin chain comprises a CH1 domain having an amino acid at position 147 (EU numbering) substituted with an amino acid selected from E or D (see WO2017055539A1, which is incorporated herein by reference in its entirety).

[0448] In some embodiments, CrossMab encoded by one or more of the polynucleotides described herein CH1-CLcv The antibody agent comprises a CH1 domain comprising one or more charge variant mutations. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a CH1 domain, and the CH1 domain comprises one or more substitutions including K147E, K213D, or a combination thereof. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a CH1 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 50 or 865. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a CH1 domain having the amino acid sequence according to SEQ ID NO: 50 or 865. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a CH1 domain comprising the G1m3 allotype. In some embodiments, CrossMab CH1-CLcv The antibody agent comprises a CH1 domain comprising the G1m17 allotype.

[0449] In some embodiments, the CrossMab encoded by one or more polynucleotides described herein CH1-CLcv antibody agent comprises a heavy chain encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences represented by SEQ ID NOs: 209-256. In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a heavy chain encoded by a nucleic acid sequence represented by any one of SEQ ID NOs: 209-256. In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a light chain encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 452 or 453. In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a light chain encoded by a nucleic acid sequence represented by SEQ ID NO: 452 or 453.

[0450] In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12) or (iv) a combination thereof. In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9) and (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12). In some embodiments, the CrossMab CH1-CLcv antibody agent comprises a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21) or (iv) a combination thereof. In some embodiments, the CrossMab CH1-CLcvThe antibody agent comprises a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GTS, SEQ ID NO: 18), and (iii) LCDR3 (QRYGGTPIT, SEQ ID NO: 21). In some embodiments, CrossMab CH1-CLcv The antibody agent comprises: (a) a heavy chain variable domain comprising (i) HCDR1 (DDPYTDDDTFTKYW, SEQ ID NO: 6), (ii) HCDR2 (ISPHFARP, SEQ ID NO: 9), (iii) HCDR3 (ARDPFGDRAPHYNYHMDV, SEQ ID NO: 12), or (iv) a combination thereof; and (b) a light chain variable domain comprising (i) LCDR1 (QGLDSSH, SEQ ID NO: 15), (ii) LCDR2 (GT...

Claims

[Claim 1] The invention described in the present specification.