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WO2024260570A1 - Anticorps codés par un acide nucléique - Google Patents

Anticorps codés par un acide nucléique Download PDF

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Publication number
WO2024260570A1
WO2024260570A1 PCT/EP2023/067201 EP2023067201W WO2024260570A1 WO 2024260570 A1 WO2024260570 A1 WO 2024260570A1 EP 2023067201 W EP2023067201 W EP 2023067201W WO 2024260570 A1 WO2024260570 A1 WO 2024260570A1
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WIPO (PCT)
Prior art keywords
nucleic acid
antibody
promoter
homodimerization
seq
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PCT/EP2023/067201
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English (en)
Inventor
Hans GROSSE
Patrick Baumhof
Christine WEINL-TENNBRUCK
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Curevac SE
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Curevac SE
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Priority to PCT/EP2023/067201 priority Critical patent/WO2024260570A1/fr
Publication of WO2024260570A1 publication Critical patent/WO2024260570A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Nucleic acid-based therapeutics provide alternative approaches to reduce costs and complexity of antibody therapies. For example, coding nucleic acids (e.g. mRNA) can be administered for delivering large amounts of antibodies in vivo.
  • nucleic acid-based therapeutics such as mRNA therapeutics have the potential to allow a variety of different antibodies to be expressed at the same time, e.g. through a single nucleic acid composition.
  • simultaneous expression of a plurality of assembled antibodies is associated with various fundamental technical problems, particularly problems associated with the correct assembly of the encoded antibodies.
  • nucleic acids encoding for multiple assembled antibodies e.g. an antibody mixture or cocktail
  • administration of one or more nucleic acids encoding for multiple assembled antibodies requires the correct assembly of all the encoded HCs and, optionally, all the encoded LCs. Since cells that get transfected with such a variety of nucleic acids can express multiple different HCs and LCs simultaneously, the correct assembly of the antibodies is more complex, and totally different to the “natural” situation where one specific B-cell produces only one type of antibody.
  • nucleic acids encoding for only two "classical” antibodies are administered by a single composition
  • such a nucleic acid composition would generate multiple unwanted mismatched by-products, e.g. by-products with mismatched HCs and/or mismatched HC/LC, and only a small portion of correctly assembled antibodies.
  • nucleic acids encoding for more than two assembled antibodies are administered. Accordingly, such an approach would eventually generate a large portion of unwanted mismatched by-products, which would then reduce or minimize the therapeutic efficacy e.g. for in vivo use.
  • these by-products could induce dramatic unwanted side-effects in a subject (e.g., in case where the misassembled antibodies show off-target binding activity).
  • WO2022023559 correct antibody assembly is facilitated by using heterodimerization promoters. Accordingly, two different HC nucleic acid sequences are needed to obtain one assembled antibody. Consequently, for the generation of antibody mixtures comprising multiple antibodies, a large number of nucleic acid sequences (e.g. constructs) is needed.
  • “about’ means, that a determinant or values may diverge by +/-1 %, +/-2%, +/-3%, +/-4%, +1-5%, +/-6%, +/- 7%, +/-8%, +1-9%, +/-10%.
  • immunoglobulin allotype refers to an antibody chain, e.g. antibody heavy chain or antibody light chain found in an individual. The term relates to the allele of the antibody chains found in the individual.
  • each immunoglobulin has unique sequences particular to the individual's genome that manifest in its constant region.
  • An “allotype” may have unique sequences particular to the individual's genome. These differences may be on amino acid level, and may be manifested in the amino acid sequence in its constant region of an antibody chain, in particular, of an antibody heavy chain or antibody light chain.
  • Gm allotypes of the IgG heavy chain
  • km allotypes of the kappa light chain
  • Gm for gamma marker
  • the allotypes G1 m, G2m, and G3m are carried by the constant region of the gammal , gamma2, and gamma3 chains, encoded by the IGHG1 , IGHG2, and IGHG3 genes, respectively.
  • Antigen The term “antigen” as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to any substance which may be recognized by components of the immune system, preferably by components of the adaptive immune system.
  • an antigen is capable of triggering an antigenspecific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • an antigen can be any target that an antibody or antigen-binding molecule is capable to bind to, e.g. a peptide, a protein, a carbohydrate, a lipid, or any combination thereof.
  • an "antibody” is a polypeptide that specifically recognizes and/or binds to a particular target.
  • target encompasses all molecules, structures, or agents that an antibody is capable to bind to.
  • the target is e.g. a peptide, a protein, a carbohydrate, a lipid, or any combination thereof.
  • Most targets of an antibody are considered to be antigens. Accordingly, the term “antibody” refers in the broadest sense to any type of antigen-binding molecule.
  • antibody may encompass various forms of antigen-binding molecules and antibodies, preferably monoclonal antibodies, including but not being limited to whole antibodies, antibodies of any (recombinant or naturally occurring) antibody format, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties of an antibody are retained.
  • antibodies are immunoglobulins or can be derived from immunoglobulins.
  • Human immunoglobulins for example, can in turn be differentiated into five main classes on the basis of their heavy chain (HC), the IgM (p), IgD (6), IgG (E), IgA (a) and IgE (E) antibodies, of those IgG antibodies making up the largest proportion.
  • Human immunoglobulins can moreover be differentiated into the isotypes K and A on the basis of their light chains.
  • IgG antibodies are typically built up by two identical light and two identical heavy chain proteins which are bonded to one another via disulfide bridges.
  • the light chain (LC) comprises the N-terminal variable domain VL (also referred to as “light chain variable region”) and the C-terminal constant domain CL (also referred to as “light chain constant region”).
  • the heavy chain (HC) of an IgG antibody can be divided into an N-terminal variable domain VH (also referred to as “heavy chain variable region”) and three constant domains CH1 , CH2 and CH3 (all three constant domains together are also referred to as “heavy chain constant region”).
  • an antibody recognizes a unique target of e.g. an antigen via its variable domains. In particular, the antibody mediates this function by binding to the target or antigen.
  • the term "antibody” refers to both, glycosylated and non-glycosylated immunoglobulins of any isotype or subclass (e.g., IgG, IgM, IgE, IgA and IgD).
  • a typical antibody is a tetramer. Each tetramer consists of two pairs of polypeptide chains, each pair having a "light chain” (LC) and a "heavy chain” (HC) as defined above.
  • antibodies include monoclonal antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetic, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, single chain antibodies, antibody derivatives, intrabodies, antibody analogues, and functional antibody fragments.
  • antibody includes, in addition to antibodies comprising two full-length heavy chains and two full- length light chains, derivatives, variants, and antibodies of any formats, which do not comprise two full-length heavy chains and/or two full-length light chains. In some instances an "antibody” may thus include fewer chains, for example a single chain or two chains only.
  • an antibody recognizes (and binds to) an antigen or a target.
  • an antibody usually comprises at least one target binding site (or “antigen binding moiety”), which is also referred to as “paratope” and which recognizes (and binds to) an epitope on the antigen or target.
  • a paratope typically comprises a set of complementary determining regions (CDRs) and usually contains parts of the light chain and parts of the heavy chain of the antibody.
  • a paratope of native IgG comprises three CDRs of the heavy chain (CDRH1 , CDRH2 and CDRH3) and three CDRs of the light chain (CDRL1 , CDRL2, and CDRL3).
  • the CDRs of an antibody are arranged in the antibody’s variable region: CDRH1 , CDRH2 and CDRH3 in the heavy chain variable region (VH) and CDRL1 , CDRL2, and CDRL3 in the light chain variable region (VL).
  • an antibody may comprise a constant region (on heavy and light chain: CH and CL, respectively).
  • the heavy chain constant region comprises three domains (CH1 , CH2 and CH3), whereas the light chain constant region comprises one domain only.
  • an antibody is typically an immunoglobulin or is derived from an immunoglobulin.
  • An antibody (or an antibody fragment) may fulfill various different functions by recognizing (and binding to) a target, e.g. an antigen, such as neutralization, agglutination, precipitation and/or complement activation. Further, antibodies may recruit one or more effector cells or molecules, e.g. immune effector cells (e.g. in the case of bispecific antibodies), or e.g. selectively engage distinct trigger molecules. Further effector functions may include fixation of complement, binding of phagocytic cells, lymphocytes, platelets, mast cells, and basophils which have immunoglobulin receptors.
  • Antibody fragments or variants, fragment or a variant of an antibody is preferably to be understood as a functional fragment or a functional variant, which may comprise at least one functional CDR of the corresponding antibody capable of recognizing (and binding to) an antigen or target.
  • antibody fragments are any antibody fragments known to a person skilled in the art, e.g. Fab, Fab', F(ab')2, Fc, Facb, pFc', Fd, und Fv fragments of the above mentioned antibodies etc.
  • a Fab (fragment antigen binding) fragment typically comprises the variable and a constant domain of a light and a heavy chain, e.g.
  • a Fab fragment thus conventionally contains the complete antigen-binding region of the original antibody and usually has the same affinity for the antigen, the immunogen or an epitope of a protein.
  • antibody fragments consisting of the minimal binding subunit of antibodies are usually known as single-chain variable fragment (scFv) and typically have excellent binding specificity and affinity fortheir ligands.
  • An scFv typically comprises the variable domain of the light and of the heavy chain, which are bonded to one another via an artificial polypeptide linker.
  • variable of an antibody has to be understood as (i) having the same or similar biological function as the corresponding reference or full length antibody or of the corresponding antibody fragment, or (ii) the same or similar activity of the corresponding reference or full length antibody or of the corresponding antibody fragment, e.g. the specific binding to particular antigens as defined herein.
  • a fragment or a variant of an antibody according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even at least 97%, with an amino acid sequence of the respective reference or full-length antibody or a fragment thereof.
  • a fragment of an antibody according to the invention may typically comprise an amino acid sequence having a sequence length of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even at least 97%, with an amino acid sequence length of the respective reference or full- length antibody or a fragment thereof.
  • Antibody light chain fragment relates to a fragment of an antibody light chain.
  • a typical antibody light chain comprises a variable domain (VL), and a constant domain (CL).
  • the term “antibody light chain fragment’ may relate to a fragment comprising or consisting of at least a fragment of VL and/or CL.
  • a fragment of a antibody light chain may be N- terminally truncated (e.g. lacking the VL domain or parts of the VL domain), or C-terminally truncated (e.g. lacking the CL domain, or parts of the CL domain), or may be N- and C-terminally truncated.
  • a fragment of an antibody light chain in the context of the invention comprises an amino acid sequence having a sequence length of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even at least 97%, with an amino acid sequence length of the respective reference or full-length antibody light chain or VL.
  • Antibody light chain variant has to be understood as (i) having the same or similar biological function as the corresponding reference or full length antibody light chain or of the corresponding antibody light chain fragment or, respectively, (ii) the same or similar activity of the corresponding reference or full length antibody light chain or of the corresponding antibody light chain fragment, e.g. the specific binding of particular antigens as defined herein.
  • a variant of an antibody light chain according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even at least 97%, with an amino acid sequence of the respective reference or full length antibody light chain or a fragment thereof, e g. a VL.
  • Antibody heavy chain fragment as used herein relates to a fragment of an antibody heavy chain.
  • Atypical antibody heavy chain comprises a variable domain (VH), and a constant region comprising three domains (CH1 , CH2 and CH3).
  • the term “antibody heavy chain fragment” may relate to a fragment comprising or consisting of at least a fragment of VH, CH1 , CH2, and/or CH3.
  • a fragment of a antibody heavy chain may be N-terminally truncated (e.g. lacking the VH domain or parts of the VH domain), or C-terminally truncated (e.g.
  • a typical fragment of an antibody heavy chain may comprise a heavy chain Fab region (comprising to VH and CH1 and a hinge region), and/or an Fc region (comprising a hinge region and CH2 and CH3). Accordingly, a typical fragment of an antibody heavy chain may comprise e.g. a VH, CH1 and a hinge region, and/or optionally an Fc region (comprising a hinge region and CH2 and CH3).
  • a fragment of an antibody heavy chain in the context of the invention comprises an amino acid sequence having a sequence length of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence length of the respective reference antibody heavy chain (e.g. full length or CH2-CH3).
  • an amino acid sequence length of the respective reference antibody heavy chain e.g. full length or CH2-CH3
  • Antibody heavy chain variant has to be understood as (i) having the same or similar biological function as the corresponding reference or full length antibody heavy chain or of the corresponding antibody heavy chain fragment or, respectively, (ii) the same or similar activity of the corresponding reference or full length antibody heavy chain or of the corresponding antibody heavy chain fragment, e.g. the specific binding of particular antigens as defined herein.
  • a variant of an antibody heavy chain according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even at least 97%, with an amino acid sequence of the respective reference or full length antibody heavy chain or a fragment thereof.
  • Artificial nucleic acid artificial DNA, artificial RNA, artificial nucleic acid sequence
  • an artificial nucleic acid may be understood as a non-natural nucleic acid molecule.
  • Such nucleic acid molecules may be non-natural due to its individual sequence (e.g. G/C content modified coding sequence, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides.
  • An artificial nucleic acid sequence may be a DNA sequence, an RNA sequence, or a hybrid-sequence comprising DNA and RNA portions.
  • an artificial nucleic acid sequence may also comprise or consist of PNA, LNA or other modified nucleotides or nucleotide analogs.
  • artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides.
  • an artificial nucleic acid is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type sequence/the naturally occurring sequence/the reference sequence by at least one nucleotide (via e.g. codon modification).
  • the term “artificial nucleic acid” is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules. Accordingly, it may relate to a plurality of essentially identical nucleic acid molecules.
  • the term “artificial nucleic acid” as used herein may for example relate to an artificial DNA or, preferably, to an artificial RNA.
  • Assembled antibody The terms “intact antibody” or “fully assembled antibody” or “assembled antibody” are used in reference to an antibody to mean that it contains two heavy chains and, optionally two light chains, optionally associated by disulfide bonds as occurs with naturally-produced antibodies. Accordingly, an “intact antibody” or “fully assembled antibody” or “assembled antibody” exerts its function, e.g. binding of at least one antigen or Fc receptor. Correct assembly depends on the desired configuration of the encoded antibody. Methods to determine assembly or misassembly of an antibody exists in the art, and may suitably be used in the context of the invention to determine the percentage of assembled antibodies and misassembled antibodies.
  • mass spectrometry can be used to determine the percentage of assembled antibodies and misassembled antibodies.
  • the nucleic acid composition encoding antibodies can be administered to cells in vitro (e.g. HeLa or HEK293T in a cell culture) using a transfection agent (e.g. lipofectamine) to allow expression and secretion of the antibodies.
  • the secreted antibodies can be purified from the cell-culture supernatant using e.g. protein A column-based FPLC.
  • the purified antibodies can be subjected to treatment with a cysteine protease that digests IgG antibodies (e.g., FabALACTICA (IgdE) (Genovis)) to yield the disulphide-bridged Fc-portion of the antibodies.
  • a cysteine protease that digests IgG antibodies
  • the disulphide-bridged Fc-portion may be deglycosylated (e.g. using PNGase).
  • the enzymatic treatment can reduce a full-length antibody (150kDa plus Glycan pattern) to an Fc portion of 50kDa without glycan pattern.
  • the samples can be analyzed using HPLC-MS to observe mass differences and to determine the ratio of assembled and misassembled antibodies. An example of such a procedure is provided in the example section.
  • the nucleic acid composition encoding antibodies may be administered to animal models e.g. to mice or rats using a suitable delivery system e.g. liposomes or LNPs.
  • Produced antibodies can be purified and analyzed using MS as described above. An example of such a procedure is provided in the example section. Additionally, MS and/or non-reducing Western blot assays can be used to analyze non-assembled antibody chains.
  • Bispecific antibody, bifunctional antibody The term “bispecific antibody” or “bifunctional antibody” relates to antibodies that comprise specificities to two antigens (bi + specific) in any of several ways: antibodies that have affinities for two antigens; antibodies that are specific to two antigens or two epitopes; or antibodies specific to two types of cell or tissues. Bispecific antibody can simultaneously bind to two different types of antigen. Accordingly, a bispecific antibody has specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. Bispecific antibodies may be in the IgG-like configuration or format.
  • This format retains the traditional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except the two Fab sites bind different antigens.
  • bispecific antibodies that lack an Fc region entirely. These include chemically linked Fabs, consisting of only the Fab regions, and various types of bivalent single-chain variable fragments (scFvs).
  • scFvs bivalent single-chain variable fragments
  • Cap 5’-cap structure, 5’-cap, cap:
  • the term “5’-cap structure” as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5’-end of a nucleic acid, e.g. an RNA or mRNA.
  • the 5’-cap structure is connected via a 5’-5’-triphosphate linkage to a nucleic acid.
  • 5’-cap structures which may be suitable in the context of the present invention are capO (methylation of the first nucleobase, e.g.
  • cap1 additional methylation of the ribose of the adjacent nucleotide of m7GpppN
  • cap2 additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN
  • cap3 additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN
  • cap4 additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN
  • ARCA anti-reverse cap analogue
  • modified ARCA e.g.
  • phosphothioate modified ARCA inosine, N1-methyl-guanosine, 2’- fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • Chimeric antibody refers to an antibody in which both chain types are chimeric as a result of antibody engineering.
  • a chimeric chain is a chain that contains a foreign variable domain (originating from a non-human species, or synthetic or engineered from any species including human) linked to a constant region of e.g. human origin.
  • the variable domain of a chimeric chain has a V region amino acid sequence which, analyzed as a whole, is closer to non-human species than to human.
  • Coding sequence/codinq region The terms “coding sequence” or “coding region” and the corresponding abbreviation “cds” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a sequence of several nucleotide triplets, which may be translated into a peptide or protein.
  • a coding sequence in the context of the present invention may be a DNA sequence, preferably an RNA sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon and which preferably terminates with a stop codon.
  • the cds of the DNA or RNA may terminate with one or two or more stop codons.
  • Codon modified coding sequence relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type (or reference) coding sequence.
  • a codon modified coding sequence in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably to optimize/modify the coding sequence for in vivo applications as outlined herein.
  • nucleic acid derived from (another) nucleic acid
  • nucleic acid which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived.
  • sequence identity is typically calculated for the same types of nucleic acids, i.e.
  • RNA sequences are converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined.
  • nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g. in order to increase RNA stability even further and/or to prolong and/or increase protein production.
  • derived from means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g.
  • Epitope The term “epitope” (also called “antigen determinant” in the art) as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to T cell epitopes and B cell epitopes.
  • T cell epitopes or parts of the antigenic peptides or proteins may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g.
  • B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • epitopes can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • expression as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to the production of a polypeptide (e.g. heavy chain or light chain of an antibody) or production of multiple polypeptides (e.g. assembled antibody), wherein said polypeptide /said multiple polypeptides are provided by a coding sequence of a nucleic acid sequence as defined herein.
  • expression of an RNA sequence refers to production of a protein via translation of the RNA into a polypeptide, or into multiple polypeptides.
  • “Expression” of a DNA sequence refers to production of a protein via transcription of the DNA into RNA and subsequent translation into protein, or into assembled multiple polypeptides.
  • expression and the term “production” may be used interchangeably herein. Further, the term “expression” preferably relates to production of a certain polypeptide (antibody chains) or multiple polypeptides (e.g. assembled antibody) upon administration of a nucleic acid (set) to a cell or an organism.
  • fragment as used throughout the present specification in the context of a nucleic acid sequence (e.g. RNA or a DNA) or an amino acid sequence may typically be a shorter portion of a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, consists of a sequence that is identical to the corresponding stretch within the full-length sequence.
  • a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e. full-length) molecule from which the fragment is derived (e.g. from an antibody chain, e.g. HC or LC).
  • entities such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e. full-length) molecule from which the fragment is derived (e.g. from an antibody chain, e.g. HC or LC).
  • fragment herein in the context of proteins or peptides may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence, N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original protein. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide.
  • heterologous refers to a sequence (e.g. RNA, DNA, amino acid). It has to be understood as a sequence that is derived from another gene, another allele, or e.g. another species or virus. Two sequences are typically understood to be “heterologous” if they are not derivable from the same gene or from the same allele. I.e., although heterologous sequences may be derivable from the same organism or virus, in nature, they do not occur in the same nucleic acid or protein.
  • Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo; or mutations introduced by antibody engineering in case of the homodimerization promoters of the present invention).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • a human antibody may be encoded by a nucleic acid sequence.
  • Humanized antibody refers to an antibody in which both chain types are humanized as a result of antibody engineering.
  • a humanized chain is typically a chain in which the complementarity determining regions (CDR) of the variable domains are foreign (originating from one species other than human, or synthetic) whereas the remainder of the chain is of human origin. Humanization assessment is based on the resulting amino acid sequence, and not on the methodology per se, which allows protocols other than grafting to be used.
  • the variable domain of a humanized chain has a V region amino acid sequence which, analyzed as a whole, is closer to human than to other species.
  • a humanized antibody may be encoded by a nucleic acid sequence of the invention.
  • Immunoglobulin isotype, isotype refers to the immunoglobulin class or sub-class, for instance IgG 1 , lgG2, lgG3, lgG4, IgD, lgA1 , lgA2, IgE, or IgM or any allotypes thereof that is encoded by heavy chain constant region genes. Further, each heavy chain isotype can be combined with either a kappa (K) or lambda (I) light chain.
  • K kappa
  • I lambda
  • the expression of a specific isotype determines the function of an antibody via the specific binding to Fc receptor molecules on different immune effector cells. Isotype expression reflects the maturation stage of a B cell. Naive B cells express IgM and IgD isotypes with unmutated variable genes, which are produced from the same initial transcript following alternative splicing.
  • Intrabody intracellularly expressed antibodies, i.e. antibodies which are coded by nucleic acids localized in the cell and are expressed there. Intrabodies can be localized and expressed at certain sites in the cell. For example, intrabodies can be expressed in the cytoplasm, the formation of disulfide bridges usually being decreased under the reducing conditions of the cytoplasm. It has been possible to demonstrate, however, that cytoplasmic intrabodies, and in particular scFv fragments, can be functional. Cytoplasmic expression opens up the possibility of also inhibiting cytoplasmic proteins. By expression of a signal peptide, intrabodies can be transported into the endoplasmic reticulum (ER) and then secreted as with regular antibodies.
  • ER endoplasmic reticulum
  • intrabodies can include full length antibodies or antibody fragments as described above.
  • Intrabodies in the context of the present invention preferably initially include full length antibodies, which are retained in the cell and not secreted from the cell (by whatever technique, e.g. retentton signal sequences etc.). However, if e.g. intracellular expression of full length antibodies is technically not possible or not appropriate, antibody fragments as described above can also be employed as intrabodies.
  • Identity (of a sequence): The term “identity” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, e.g. nucleic acid sequences or amino acid (aa) sequences as defined herein, preferably the aa sequences encoded by the nucleic acid sequence as defined herein or the aa sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence.
  • a position in the first sequence is occupied by the same residue as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using an algorithm, e.g. an algorithm integrated in the BLAST program.
  • mixed isotype refers to Fc region of an immunoglobulin generated by combining structural features of one isotype with the analogous region from another isotype thereby generating a hybrid isotype.
  • a mixed isotype may comprise an Fc region having a sequence comprised of two or more isotypes selected from the following lgG1 , lgG2, lgG3, lgG4, IgD, lgA1 , lgA2, IgE, or IgM thereby generating combinations such as e.g. lgG1/lgG3, lgG1/lgG4, lgG2/lgG3 or lgG2/lgG4.
  • Mixture of different antibodies denotes a composition comprising different antibody molecules which may differ with respect to their amino acid sequence. Accordingly, different antibodies in a mixture (e.g. at least two) represent different antibody species. Identical antibodies in the mixture belong to the same antibody molecule species. Antibodies of different species differ with respect to their sequence and/or their structure. Hence, a “species” denotes a group of essentially identical antibody molecules.
  • Monospecific antibody relates to antibodies whose specificity to antigens is singular (mono- + specific) in any of several ways: antibodies that all have affinity for the same antigen; antibodies that are specific to one antigen or one epitope; or antibodies specific to one type of cell or tissue.
  • the terms “monospecific” and “monovalent” may in some cases be used interchangeably; both can indicate specificity to one antigen, one epitope, or one cell type.
  • a monospecific antibody suitably comprises two essentially identical target binding sites.
  • multispecific antibody relates to antibodies that comprise specificities to multiple antigens (multi- + specific) in any of several ways: antibodies that have affinities for multiple antigens; antibodies that are specific to multiple antigens or multiple epitopes; or antibodies specific to multiple types of cell or tissues.
  • multispecific and multivalent may in some cases be used interchangeably; both can indicate specificity to multiple antigens, multiple epitopes, or multiple cell types.
  • a multispecific antibody would comprise at least two different target binding sites.
  • hexaspecific antibody relates to antibodies that comprise specificities to five or six antigens in any of several ways: antibodies that have affinities for five or six antigens; antibodies that are specific to five or six antigens or five or six epitopes; or antibodies specific to five or six types of cell or tissues.
  • Poly(A) sequence, polv(A) tail, 3’-poly(A) tail The terms “poly(A) sequence”, “poly(A) tail” or “3’-poly(A) tail” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to be a sequence of adenosine nucleotides, typically located at the 3'-end of a linear nucleic acid (e.g. mRNA), of up to about 1000 adenosine nucleotides.
  • a linear nucleic acid e.g. mRNA
  • Poly(C) sequence, polv(C) tail, 3’-polv(C) tail The term “poly(C) sequence” as used herein is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides.
  • a poly(C) sequence may comprise about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides, e.g. 30 cytosines.
  • RNA in vitro transcription or “in vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system in vitro.
  • RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which is typically a linear DNA template (e.g. linearized plasmid DNA or PCR product).
  • the promoter for controlling RNA in vitro transcription can be any promoter for any DNA- dependent RNA polymerase.
  • DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases.
  • the DNA template is typically linearized with a suitable restriction enzyme before it is subjected to RNA in vitro transcription.
  • Reagents typically used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase; ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein; optionally, modified nucleotides as defined herein; a DNA- dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g.
  • RNA polymerase T7, T3, SP6, or Syn5 RNA polymerase
  • RNase ribonuclease
  • MgCI2 MgCI2
  • TIS or HEPES buffer
  • suitable pH value which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine.
  • Stabilized nucleic acid refers to “stabilized RNA” or “stabilized DNA” and is intended to comprise nucleic acid that is modified such, e.g. that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by exo- or endonuclease degradation, compared to an nucleic acid without such modification.
  • a stabilized nucleic acid e.g. RNA or DNA
  • a stabilized nucleic acid in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell.
  • the stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., e.g., for storage.
  • Single domain antibody also known as nanobody®, is an antibody fragment consisting of a single monomeric variable antibody chain or domain. Like a whole antibody, a single domain antibody is able to bind selectively to a specific antigen or target.
  • the first single-domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called VHH fragments.
  • Cartilaginous fishes also have heavychain antibodies (IgNAR, “immunoglobulin new antigen receptor”), from which single-domain antibodies called VNAR fragments can be obtained.
  • IgNAR immunoglobulin new antigen receptor
  • An alternative approach is to split the dimeric variable domains from common IgG into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to bind specifically to target epitopes.
  • Single chain antibody means an “antibody” that consists of only one polypepide chain (i.e. intrinsically does not require antibody chain assembly).
  • Typical examples of such antibodies are single-chain antibody fragments (scAb; typically VH-VL-CL) or single-chain variable fragments (scFv).
  • scAb single-chain antibody fragments
  • scFv single-chain variable fragments
  • the latter typically relate to fusion proteins of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, typically connected with a short linker peptide of e.g. ten to about 25 amino acids.
  • the linker may for example be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
  • This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs.
  • bispecific tandem di-scFvs often referred to as bi-specific T-cell engagers (BiTE antibody constructs). More single-chain antibody formats may be found e.g. in Brinkmann & Kontermann (2017; mAbs; PMID 28071970; e.g. Figure 2).
  • scFv-Fc and scFab-IgG are not single chain antibodies within the meaning of the present invention, but rather assembled antibodies.
  • Tetraspecific antibody, tetrafunctional antibody The term “tetraspecific antibody” or “tetrafunctional antibody” relates to antibodies that comprise specificities to four antigens (tetra- + specific) in any of several ways: antibodies that have affinities for four antigens; antibodies that are specific to four antigens or four epitopes; or antibodies specific to four types of cell or tissues.
  • the terms “tetraspecific” and “tetravalent” may in some cases be used interchangeably; both can indicate specificity to four antigens, four epitopes, or four cell types.
  • a tetraspecific antibody would comprise at least two different target binding sites and at least two further target binding sites.
  • Trispecific antibody trifunctional antibody: The term “trispecific antibody” or “trifunctional antibody” relates to antibodies that comprise specificities to three antigens (tri + specific) in any of several ways: antibodies that have affinities for three antigens; antibodies that are specific to three antigens or three epitopes; or antibodies specific to three types of cell or tissues.
  • the terms “trispecific” and “trivalent” may in some cases be used interchangeably; both can indicate specificity to three antigens, three epitopes, or three cell types. In the context of the invention, a trispecific antibody would comprise at least three different target binding sites.
  • Untranslated i, UTR, UTR element The term “untranslated region” or “UTR” or “UTR element’ will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a part of a nucleic acid typically located 5’ or 3’ of a coding sequence.
  • An UTR is not translated into protein.
  • An UTR may be part of a nucleic acid, e.g. a DNA or an RNA.
  • An UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements etc.
  • 5’-untranslated region, 5’-UTR, 5-UTR element The terms “5’-untranslated region” or “5-UTR” or “5-UTR element’ will be recognized and understood by the person of ordinary skill in the art, and are e.g. intended to refer to a part of a nucleic acid molecule located 5’ (i.e. “upstream”) of a coding sequence and which is not translated into protein.
  • a 5’- UTR may be part of a nucleic acid located 5’ of the coding sequence.
  • a 5-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence.
  • a 5-UTR may comprise elements for controlling gene expression, also called regulatory elements.
  • Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.
  • the 5-UTR may be post-transcriptionally modified, e.g. by enzymatic or post-transcriptional addition of a 5’-cap structure in e.g. RNA.
  • Variant of a sequence:
  • the term “variant” as used herein in the context of a nucleic acid sequence is e.g. intended to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence.
  • a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
  • a variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from.
  • the variant is preferably a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
  • a “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.
  • variant as used herein in the context of proteins or peptides is e.g.
  • proteins or peptide variants having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific property.
  • “Variants” of proteins or peptides as defined herein may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence.
  • a “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide.
  • a variant of a protein comprises a functional variant of the protein, which means that the variant exerts the same effect or functionality or at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the effect or functionality as the protein it is derived from.
  • the present invention relates inter alia to a nucleic acid for expression of an antibody comprising homodimeric antibody heavy chain (HC) elements in a cell or subject, wherein the nucleic acid comprises at least one coding sequence encoding at least one antibody HC element that comprises a homodimerization promoter. Further, the invention relates to a nucleic acid set, an antibody encoded by the nucleic acid, a composition, a combination and kit or kit of parts in this context. Additionally, first and second medical uses, methods of treating or preventing diseases, disorders or conditions, methods of expressing and producing are provided.
  • HC homodimeric antibody heavy chain
  • the invention relates to a nucleic acid cassette encoding a homodimerization promoter, a homodimerization promoter as such, and methods of identifying and selecting suitable homodimerization promoters for expression of one or more nucleic acid-encoded antibodies.
  • Nucleic acid-based therapeutics such as mRNA therapeutics have the potential to allow a variety of different antibodies to be expressed at the same time, e.g. through a single nucleic acid composition.
  • simultaneous expression of a plurality of assembled antibodies is associated with various fundamental technical problems, particularly problems associated with the correct assembly of the encoded antibodies, as further outlined below.
  • nucleic acids encoding for multiple assembled antibodies e.g. an IgG antibody cocktail
  • administration of one or more nucleic acids encoding for multiple assembled antibodies requires the correct assembly of all the encoded HCs and, optionally, all the encoded LCs of each antibody.
  • nucleic acids encoding for two IgG antibodies e.g. Antibody 1 , Antibody 2
  • only a minority of correctly assembled antibodies Antibody 1 : LC1 -HC1 -HC1 -LC1 ; Antibody 2: LC2-HC2-HC2-LC2
  • a majority of unwanted mismatched byproducts e.g.
  • the present invention is, in part, based on the surprising finding that the simultaneous production and correct assembly of a plurality of assembled antibodies can be accomplished by delivering one or more nucleic acids encoding said plurality of assembled antibodies, wherein at least one nucleic acid comprises at least one coding sequence encoding at least one antibody HC element that comprises a homodimerization promoter.
  • the inventive approach is supported by experiments provided in the Example section where the inventors identified suitable homodimerization promoters that allow the expression of a mixture of correctly assembled antibodies in a cell or subject.
  • An exemplary illustration how an homodimerization promoter of the invention can support assembly and, at the same time, can prevent misassembly is shown in Figures 1 and 2.
  • RNA-based therapeutics encoding a mixture of assembled antibodies
  • the inventors have successfully demonstrated that antibody mixtures can be delivered by nucleic acid sequences and produced upon administration, which makes it possible to eliminate the highly expensive and time-consuming recombinant antibody manufacturing process.
  • the antibody mixtures produced according to the teaching of the present invention show a high percentage of correctly assembled antibody (species), which is a prerequisite for therapeutic use of nucleic acid-based therapeutics encoding antibody mixtures.
  • mispairing to other antibody heavy chains could be prevented (e.g. to heavy chains that do not comprise assembly promoters, e.g. wild type (unmodified) heavy chains).
  • the present invention relates to a nucleic acid forexpression of an antibody comprising homodimeric antibody HC elements in a cell or subject, wherein the nucleic acid comprises at least one coding sequence encoding at least one antibody HC element that comprises a homodimerization promoter.
  • the present invention relates to a nucleic acid set for expression of an antibody in a cell or subject, wherein the nucleic acid set comprises a first nucleic acid as defined in the context of the first aspect and a second nucleic acid comprising at least one coding sequence encoding at least one antibody LC element.
  • the present invention relates to an antibody encoded by any nucleic acid as defined in the first aspect or by any nucleic acid set as defined in the second aspect.
  • the invention in a fourth aspect, relates to a composition comprising at least one nucleic acid as defined in the first aspect and/or at least one nucleic acid set as defined in the second aspect.
  • the invention relates to a combination of at least one nucleic acid as defined in the first aspect; and/or at least one nucleic acid set as defined in the second aspect; and/or at least one antibody as defined in the third aspect; and/or at least one composition as defined in the fourth aspect.
  • the invention in a sixth aspect, relates to a kit or kit of parts, comprising at least one nucleic acid of the first aspect, at least one nucleic acid set of the second aspect, at least one antibody of the third aspect, at least one composition of the fourth aspect, or at least one combination of the fifth aspect, optionally comprising at least one liquid vehicle for solubilising, and, optionally, technical instructions providing information on administration and dosage of the kit components.
  • the invention relates to first and second medical uses as well as methods of treatments.
  • the invention relates to methods of expressing at least one, preferably at least two nucleic acid- encoded antibodies in an organ or tissue or a subject, and to methods of producing at least one, preferably at least two nucleic acid-encoded antibodies in vitro, in situ, or ex vivo.
  • the invention relates to a nucleic acid cassette encoding a homodimerization promoter, to a homodimerization promoter as such, and methods of identifying and selecting suitable homodimerization promoters for expression of one or more nucleic acid-encoded antibodies.
  • the present invention relates inter alia to a nucleic acid for expression of an antibody that comprises homodimeric antibody heavy chains (HC) or HC elements in a cell or subject.
  • HC homodimeric antibody heavy chains
  • nucleic acid of the first aspect may likewise be applied to the nucleic acid set of the second aspect, the antibody of the third aspect, the composition of the fourth aspect, the combination of the fifth aspect, and any further aspect of the invention.
  • the nucleic acid comprises at least one coding sequence encoding at least one antibody heavy chain (HC) or HC element that comprises a homodimerization promoter.
  • HC antibody heavy chain
  • antibody HC element relates to a polypeptide element selected or derived from an antibody heavy chain. Such an “antibody HC element” may be a fragment of an antibody heavy chain or may be a full length antibody heavy chain.
  • An antibody in the context of the invention may be without being limited thereto, any type of a monospecific antibody, a bispecific antibody, muttispecific antibody, a chimeric antibody, a humanized or human antibody, a heavy-chain-only antibody, a synthetic antibody, an antibody mimetic, an antibody fusion protein, an antibody conjugate, an antibody derivative, an intrabody, or any antibody analogue or functional antibody fragment thereof.
  • Antibodies encoded by the nucleic acid can be chosen from all antibodies or antibody fragments as defined herein, in particular antibodies or antibody fragments which are or which can be employed for (any) therapeutic or for (any) diagnostic or for (any) research purposes or have been found or are employed in a particular disease, e.g. cancer diseases, infectious diseases, autoimmune diseases, inflammatory diseases etc.
  • the nucleic acid of the invention is for expression of an antibody comprising homodimeric antibody HC elements.
  • the term “homodimeric” antibody HC elements as used herein has to be understood as two HC elements with the same amino acid sequence that assemble to form HC element homodimers (i.e. homodimerize) upon nucleic acid expression.
  • the nucleic acid in the context of the invention comprises at least one coding sequence encoding at least one antibody HC element that comprises a homodimerization promoter.
  • the homodimerization promoter supports, promotes, forces, or directs the assembly of two expressed antibody HC elements comprising the same homodimerization promoter.
  • the homodimerization promoter prevents or reduces assembly with an antibody HC element that does not comprise the same or any homodimerization promoter.
  • homodimerization promote as used throughout the invention relates to at least one moiety (e.g. an amino acid or an assembly element) that promotes, supports, forces, or directs the correct assembly of at least two expressed antibody heavy chains (herein, provided by the nucleic acid sequence). Further, a “homodimerization promote preferably prevents or reduces misassembly.
  • a moiety is typically at least one amino acid substitution capable of promoting, supporting, forcing, or directing a certain assembly of two antibody heavy chains.
  • an amino acid substitution is a substitution that does not occur naturally, suitably, a substitution that does not occur naturally in human antibody heavy chains.
  • a “homodimerization promoter may comprise one or more “assembly elements" as further specified herein.
  • a “homodimerization promoter may be located on an antibody heavy chain to promote, support, force, or direct correct assembly between two same heavy chains, e.g. to promote, support, force, or direct e.g. a homodimerization of HCs.
  • a “homodimerization promoter promotes, supports, forces, or directs (correct) assembly of at least two expressed antibody heavy chains wherein said at least two antibody heavy chains have the same homodimerization promoter.
  • a suitable “homodimerization promoter may promote, support, force, or direct (correct) assembly of at least two antibody heavy chains while, at the same time, avoiding assembly to other antibody polypeptide chains lacking the "homodimerization promoter or comprising a different “homodimerization promoter.
  • said at least one moiety of the “homodimerization promoter (e.g. at least one amino acid or at least one assembly element) is encoded by the at least one coding sequence of the nucleic acid.
  • antibody heavy chains comprising an “homodimerization promoter may show an increased occurrence of correctly assembled antibody heavy chains under certain conditions, compared to naturally occurring or non-modified antibody chains lacking such an “homodimerization promoter.
  • An increased occurrence of correctly assembled antibody heavy chains is suitably observed also in the presence of other antibody polypeptide chains (e.g. lacking a homodimerization promoter) that can be provided e.g. via additional nucleic acid sequences in the composition (more details provided in e.g. the fourth aspect).
  • HCs and LCs are co-translationally translocated into the ER of a B-cell, and folding begins before the polypeptide chains are completely translated.
  • Most IgGs assemble first as HC dimers to which LCs are added covalently via a disulphide bond between the CL and CH1 domains.
  • Heavy chain assembly is typically induced by the last domain (the C-terminal domain) of the constant region, e.g. the CH3 domain in case of IgG 1. Interaction of two heavy chains involves about 16-29 amino acid residues at the interface of the two heavy chains (CH3-CH3 interface).
  • a typical antibody heavy chain comprises a natural antibody chain assembly sequence, forming a CH3-CH3 interface that mediates assembly. It has to be emphasized that such naturally occurring antibody chain assembly interfaces are not comprised by the term “homodimerization promoter” as used herein.
  • a “homodimerization promoter” may be derived from any naturally occurring antibody heavy chain assembly sequence, wherein at least one amino acid residue is mutated/changed/substituted to e.g. another amino acid residue. Accordingly, the term “homodimerization promoted’ has to be understood as “non-naturally occurring” in terms of the amino acid sequence or the position in an antibody chain (specifically, “non-naturally occurring” has to be understood in comparison to wild-type or reference antibody heavy chains, e.g. of a given species, antibody isotype, subclass or allotype).
  • the at least one “homodimerization promoter” may be located on an antibody heavy chain (that is encoded by the coding sequence).
  • an “homodimerization promoter” allows for the formation of an assembled antibody by providing one HC nucleic acid sequence. This is in contrast to the technical solution provided in WO2022023559, where correct antibody assembly is facilitated by using heterodimerization promoters. Accordingly, in WO2022023559, two different HC nucleic acid sequences are needed to obtain one assembled antibody.
  • nucleic acid of the invention advantageous features and embodiments of the nucleic acid of the invention are defined and described.
  • advantageous embodiments and features of the encoded antibody, the encoded antibody HC element, the homodimerization promoter (and its respective assembly elements) and general nucleic acid features and embodiments are defined and described.
  • all embodiments and features of said nucleic acid provided in the context of the first aspect (“the nucleic acid”) are likewise applicable to nucleic acids provided in the context of the second aspect (‘the nucleic acid set’), the fourth aspect (“the composition”), the fifth aspect (“the combination”), the sixth aspect (‘kit or kit of parts”) or to any further aspect described herein (e.g. “medical use”, “method of treatment”, “method of expressing antibodies”, etc.).
  • the nucleic acid comprises at least one coding sequence encoding at least one antibody heavy chain or HC element that comprises a homodimerization promoter, wherein the homodimerization promoter supports, promotes, forces, or directs the assembly of two antibody heavy chains or HC elements comprising the same homodimerization promoter.
  • the homodimerization promoter prevents or reduces assembly with an antibody heavy chain or HC element that does not comprise the same homodimerization promoter.
  • the homodimerization promoter prevents or reduces assembly of HCs to a wild-type (unmodified) antibody heavy chain, preferably to a wild-type (unmodified) antibody heavy chain selected or derived from a human. This is particularly advantageous in the context of in vivo applications, as a mispairing to endogenous antibody heavy chains (or other heavy chains provided by a different nucleic acid sequence) can be prevented which reduces sideeffects and increases efficacy for medical applications.
  • the homodimerization promoter prevents or reduces assembly with such a wildtype (or reference) HC element or HC.
  • the nucleic acid of the invention is administered together with a nucleic acid that encodes a different dimerization promoter (e.g. another type of homodimerization promoter or a heterodimerization promoter)
  • the homodimerization promoter prevents or reduces assembly with a HC element or HC comprising such a different dimerization promoter.
  • the homodimerization promoter prevents or reduces assembly with an antibody heavy chain or HC element that does not comprise a homodimerization promoter and/or that is a wild-type (unmodified) heavy chain or HC element.
  • the homodimerization promoter prevents or reduces assembly with an antibody heavy chain or HC element that does not comprise the same homodimerization promoter. In preferred embodiments, the homodimerization promoter prevents or reduces assembly with an antibody heavy chain or HC element that does not comprise a homodimerization promoter and/or that is a wild-type (unmodified) HC element. Preventing or reducing misassembly is particularly advantageous in the context of in vivo applications, e.g. to ensure efficacy and to reduce side-effects for medical applications.
  • the homodimerization promoter comprises at least one amino acid residue, preferably at least two amino acid residues, that do not occur naturally at the respective positions and/or at least one amino acid sequence that does not occur naturally.
  • Non-naturally occurring in terms of the amino acid sequence or the position in an antibody chain has to be understood in comparison to wild-type or reference antibody heavy chains, e.g. of a given species, antibody isotype, subclass and/or allotype.
  • the (produced) antibody comprises homodimeric antibody heavy chains or HC elements. Accordingly, the provision (e.g. the administration to a cell or subject) of a nucleic acid encoding at least one antibody HC element that comprises a homodimerization promoter suitably leads to a translation or production of the encoded HC or HC element and to an assembly of the antibody HC elements. Accordingly, the (produced) antibody comprises homodimeric antibody HC elements which preferably means that the antibody comprises homodimeric heavy chains. In preferred embodiments, the antibody is derived or selected from a monoclonal antibody or a fragment or variant thereof.
  • the antibody is derived or selected from a chimeric antibody, humanized antibody, human antibody, or a fragment or variant of any of these.
  • the antibody is derived or selected from an IgG, IgD, IgA, IgE, IgM, IgNAR, hcIgG, or a fragment or variant of any of these.
  • the antibody is derived or selected from an IgG or a fragment or variant thereof.
  • the antibody is derived or selected from an lgG1 , lgG2, lgG3, lgG4, IgD, lgA1 , lgA2, IgE, IgM, or a fragment or variant of any of these.
  • the antibody is derived or selected from IgG 1 or lgG3, or a fragment or variant of any of these.
  • the antibody is derived or selected from IgG 1 or a fragment or variant thereof.
  • the antibody is derived or selected from a full-length antibody, IgG-HC-scFv, IgG-dAb, IgG- taFv, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CaCP) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab- lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3), IgG-scFv(LC), scFv(LC)-lgG, dAb-IgG, DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody, Di-diabody-Fc, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb
  • the antibody is derived or selected from from a full-length, scFv-Fc or scFab-IgG antibody, in most preferred embodiments from a scFv-Fc antibody.
  • scFab-IgG antibody in this context preferably means an assembled homodimer of two chains with following sequence from N- to C-terminus: VL-CL-[preferab/y linker]-VH-CH1-hinge-CH2-CH3.
  • scFv-Fc antibody in this context may mean an assembled homodimer of two chains with following sequence from N- to C-terminus: VL-[preferab/y linker]-VH-hinge-CH2-CH3 (VL-VH orientation).
  • scFv-Fc antibody in this context may mean an assembled homodimer of two chains with following sequence from N- to C-terminus: H-[preferably linker]-VL-hinge-CH2-CH3 (VH-VL orientation). All of these fusion constructs have the advantage that they only need assembly of the heavy chains or HC elements, i.e. no further assembly of the HC and LC.
  • the nucleic acid encodes at least one antibody or a fragment or variant of an antibody, wherein the at least one antibody or antibody fragment or variant thereof is derived or selected from a scFv-Fc or scFab-Fc antibody. Accordingly, in preferred embodiments, the nucleic acid comprises at least one coding sequence encoding at least one single chain variable fragment (or a fragment or variant thereof). In preferred embodiments, the nucleic acid encodes at least one antibody or a fragment or variant of an antibody, wherein the antibody or antibody fragment specifically recognizes and/or binds to at least one target. In preferred embodiments, a target may be selected from at least one epitope or at least one antigen.
  • the at least one target is selected from at least one tumor antigen or epitope, at least one antigen or epitope of a pathogen, at least one viral antigen or epitope, at least one bacterial antigen or epitope, at least one protozoan antigen or epitope, at least one antigen or epitope of a cellular signalling molecule, at least one antigen or epitope of a component of the immune system, at least one antigen or epitope of an intracellular protein, or any combination thereof.
  • the at least one antibody or antibody fragment specifically recognizes and/or binds to at least one target selected from at least one antigen or epitope of a pathogen, preferably a virus or a bacterium.
  • the antibody is derived or selected from a monospecific antibody, multispecrfic antibody, or a fragment or variant of any of these.
  • the multispecific antibody is derived or selected from a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific antibody, or a fragment or variant of any of these.
  • the antibody heavy chain or HC element is derived or selected from an antibody HC, or a fragment or variant thereof, wherein the antibody heavy chain or HC element is derived or selected from an antibody as defined herein.
  • the antibody heavy chain or HC element is derived or selected from an antibody HC, or a fragment or variant thereof, selected from lgG1 , lgG2, lgG3, lgG4, IgD, lgA1 , lgA2, IgE, or IgM, or an allotype, an isotype, or mixed isotype or a fragment or variant of any of these.
  • the at least one antibody heavy chain or HC element is derived or selected from antibody heavy chains selected from lgG1 or lgG3.
  • the at least one antibody heavy chain or HC element comprises at least a CH3 domain, or a fragment or variant thereof.
  • the at least one antibody heavy chain or HC element is derived or selected from an antibody HC variant comprising a VH, CH2, CH3 domain and a hinge region, but lacking a CH1 domain.
  • the heavy chain (HC) of an IgG antibody can be divided into an N-terminal variable domain VH (also referred to as “heavy chain variable region”) and three constant domains CH1 , CH2 and CH3 (all three constant domains together are also referred to as “heavy chain constant region”).
  • VH also referred to as “heavy chain variable region”
  • CH1 , CH2 and CH3 all three constant domains together are also referred to as “heavy chain constant region”.
  • the at least one antibody heavy chain or HC element comprises aVH, CH2, CH3 but lacking a CH1 domain, e.g. for scFv-Fc or scFab-IgG.
  • the at least one antibody heavy chain or HC element is derived or selected from a full length antibody HC. Accordingly, the at least one antibody heavy chain or HC element comprises VH, CH1 , CH2, CH3.
  • the at least one antibody heavy chain or HC element is derived or selected from lgG1 or lgG3, preferably from human lgG1 or lgG3 or an allotype or an isotype thereof.
  • the at least one antibody that is provided by the nucleic acid is an IgG or is derived from an IgG.
  • An antibody that is “derived from an IgG” has to be understood as an antibody that comprises two heavy chains (derived from an IgG heavy chain).
  • an antibody that is “derived from an IgG” additionally comprises at least a portion of a light chain, preferably at least two light chains.
  • specific allotypes of heavy chains are selected to e.g. improve protein half life e.g. after expression of the antibody in a cell or a subject (e.g., upon administration of the composition).
  • specific IgG heavy chains show improved or increase FcRn recycling which leads to longer half-life of the protein such as described by Temant et al. (2016; Journal of Immunology; PMID 26685205), which disclosure is herewith incorporated in its entirety.
  • Most Gm allotypes are located in the Fc-region (CH2 or CH3) of antibodies, with the exception of G1 m3 which is linked to amino acid changes in the CH1 -region: expressing Arg rather than Lys at position 120.
  • G1m3 also expresses unique amino acids at positions 356 (Glu) and 358 (Met) in CH3 as opposed to Asp/Leu common to all G1 ml allotypes.
  • the at least one antibody heavy chain or HC element is derived or selected from heavy chain allotype G1m17.
  • Allotype G1m17 corresponds to the gene IGHG1 CH1 [K120, a359] according to the IMGT unique numbering for C-DOMAIN (Exon numbering 97, Eu numbering 214).
  • the allotype G1m17 (CH1 K120) is found on alleles IGHGV01, IGHGV02, IGHGV04, IGHG1*05, IGHG1*05p, IGHG1*06p and IGHG1*07p.
  • G1m17,1 (K120;D12/L14) and/or G1m17,-1 are selected as suitable allotypes.
  • the at least one antibody heavy chain or HC element is derived or selected from heavy chain allotype G1 ml .
  • the allotype G1 ml corresponds to the gene IGHG1 CH3 [D12, t36; L14, c40] according to the IMGT unique numbering for C-DOMAIN (Exon numbering 16 and 18, Eu numbering 356 and 358).
  • the allotype G1m1 (CH3 D12, L14) is found on alleles IGHGV01, IGHG1*02, IGHG1*04, IGHGV05, IGHG1*05p IGHG1*06p, IGHG1*07p and IGHG1*08p-
  • the at least one antibody heavy chain or HC element is derived or selected from lgG1 allotype G1m3,1 (R120, D12/L14), e.g. for full-length orscFab-IgG antibodies.
  • G1m3,1 is suitably used as G1m3,1 shows a prolonged protein half-life (Temant et al. 2016; Journal of Immunology; PMID 26685205).
  • the allotype is preferably G1m1 (D12/L14).
  • G1m3,1 is suitably used as G1m3,1 shows a prolonged protein half-life (Temant et al. 2016; Journal of Immunology; PMID 26685205).
  • Binding energy can be determined using in silico models with a protein design software such as CCG’s MOE. Intended changes are assessed via introduction of mutations (residue scan) in an energy-minimized dimeric Fc-model (based on available structural data). Generated models comprise two explicitly modelled chains, despite the symmetry of the interface. This allows further sampling by having two separate replicates to assess. Calculated energy values allow to judge the impact of the mutations respective to the input/wt model and are provided in kcal/mol.
  • the assembly of homodimeric antibody heavy chains or HC elements comprising the homodimerization promoter is preferably characterized by a more favourable (in particular a lower) binding energy than the assembly of corresponding non-modified (wildtype) heavy chains or HC elements.
  • the assembly of non-desired hetero-pairs is energetically unfavoured.
  • the explicit modelling of the two chains allows independent assessment in both directions (AG HC r/homo-dimer and AG HChomo/wr- dimer). To obtain most reliable results, a mean value of mispairing energy between the chain is generated.
  • the term “energetically unfavoured” means that the calculated energy difference caused by introduction of the homodimerization promoter in a hetero interface is associated with an energy loss (positive AAG).
  • the homodimerization promoter results in a calculated negative (i.e. lower than 0 kcal/mol) AAG, preferably lower than -1 kcal/mol, -2 kcal/mol.
  • the homodimerization promoter results in a calculated positive (i.e. greater than 0 kcal/mol) mean MG, preferably greater than 1 kcal/mol, 2 kcal/mol, 3 kcal/mol.
  • the assembly of the desired homodimeric antibody heavy chains or HC elements comprising the homodimerization promoter is more favourable over the undesired hetero-pairs with non-modified (wildtype) heavy chains or HC elements.
  • the difference between the mean unfavoured hetero-assembly (HCwr/homo and HChomo/wr) and the desired homo-assembly is as positive as possible.
  • the term “more favourable” means that the calculated energy difference caused by introduction of the assembly promoter is lower when the two chains match (introduction of homodimerization promoter in both chains) compared to the hetero pair with an unmodified chain (introduction of homodimerization in one chain only).
  • MG mean MG (as in Formula 2) - MG (as in Formula 1).
  • values for this calculated difference are >0 kcal/mol, >0.5 kcal/mol, >1.5 kcal/mol, or > 4 kcal/mol, preferably 0.5-5 kcal/mol or 1.5-5 kcal/mol.
  • MG calculated according to Formula 1 is not greater than X, preferably negative (i.e. lower than 0 kcal/mol).
  • mean MG calculated according to Formula 2 is preferably positive (i.e. greater than 0 kcal/mol).
  • MG calculated according to Formula 3 is preferably positive (i.e. greater than 0 kcal/mol, preferably >0-1 , >1 -2, >2 kcal/mol).
  • the homodimerization promoter is located in the constant region of the at least one antibody heavy chain or HC element.
  • the term “constant region of antibody heavy chain” has to be understood as foe region of an antibody heavy chain that does (typically) not contribute to target (e.g. antigen or epitope) binding.
  • the constant region of an antibody heavy chain comprises at least one of a CH1 , a CH2, and/or a CH3 domain, or a fragment or a variant thereof (as well as a hinge region).
  • the constant region of an antibody heavy chain comprises at least a CH3 domain, or a fragment or a variant thereof.
  • the constant region of an antibody heavy chain comprises a hinge region, a CH2 and a CH3 domain, optionally additionally a CH1 domain (e.g. for fall-length antibodies or scFab-IgG).
  • the homodimerization promoter is located in the Fc region of the at least one antibody heavy chain or HC element.
  • the fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate and/or interact with the immune system.
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains (CH2 and CH3).
  • IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide.
  • the homodimerization promoter is located in a CH3 (e.g. in case of IgG, IgA or IgD) or CH4 (e.g. in case of IgM or IgE) domain of the at least one antibody heavy chain or HC element.
  • heavy chain assembly is typically mediated by the last domain (the C-terminal domain) of the constant region, i.e. CH3 (IgG, IgA, IgD assembly) or CH4 (IgE, IgM assembly).
  • CH3 IgG, IgA, IgD assembly
  • CH4 IgE, IgM assembly
  • interaction of two IgG heavy chains involves about 14, 15, 16, 17, or 18 amino acid residues at the interface of the two heavy chains (CH3-CH3 interface).
  • Said about sixteen amino acid residues on each CH3 domain are typically located on four anti-parallel p-strands.
  • disulphide bonds in the hinge region connect the two heavy chains to form a HC-HC homodimer.
  • a typical antibody heavy chain comprises a natural antibody heavy chain assembly sequence interface, forming a CH3-CH3 interface (IgG, IgA, IgD) that mediates assembly. It has to be emphasized that such naturally occurring antibody heavy chain assembly interfaces are not comprised by the term “homodimerization promoter” as used herein.
  • the homodimerization promoter is located in a CH3 domain of the at least one antibody heavy chain or HC element wherein the at least one antibody heavy chain or HC element is preferably selected or derived or selected from IgG.
  • a homodimerization promoter may be located in a CH3 domain, or in a fragment or a variant of a CH3 domain, wherein the CH3 domain comprises at least one mutation or at least one amino acid substitution.
  • the CH3 domain may comprise at least one mutation or at least one amino acid substitution compared to a naturally occurring or reference CH3 domain.
  • the homodimerization promoter is located in in a region or sequence that is able to form a parallel C2-symmetry interface. In particularly preferred embodiments, the homodimerization promoter is located in in a region or sequence that corresponds to the assembly interface of wildtype (unmodified) antibody HCs.
  • the term “C2 symmetry interface” relates to the interface region of proteins that form a homodimer. Such a “C2 symmetry interface” may comprise parallel regions and/or antiparallel regions. It is preferred in the context of the invention that the homodimerization promoter is located in a “C2 symmetry interface”.
  • the homodimerization promoter is located in a CH3-CH3 assembly interface. Accordingly, the homodimerization promoter may be located in a CH3 domain, preferably in the region or the amino acid sequence that generates/defines a CH3-CH3 interface between two antibody heavy chains.
  • the CH3 domain of human IgG ranges from amino acid 342 to amino acid 446 (numbering according to EU numbering as derived from Edelman, Gerald M., et al. "The covalent structure of an entire yG immunoglobulin molecule.” Proceedings of the National Academy of Sciences 63.1 (1969): 78-85).
  • Atypical CH3-CH3 interface in e.g.
  • an lgG1 heavy chain is located in an amino acid element ranging from amino acid position aa E345 to amino acid position aa L410 (numbering according to EU numbering).
  • Contact residues in the CH3-CH3 interface may include residues e.g. at positions 347, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 360, 364, 366, 368, 370, 390, 392, 394, 395, 397, 399, 400, 405, 407, 409 according to the EU numbering system. Further contact residues may be located at position 439 or 441.
  • CH3 domain of one heavy chain typically interacts in such a interface region with a second heavy chain to allow formation of a CH3-CH3 interface.
  • a representative amino acid sequence stretch (spanning from aa E345 to amino acid position aa L410) involved in CH3-CH3 assembly is provided in SEQ ID NO: 81 of WO2022023559. Accordingly, all homodimerization promoter elements and all amino acid substitutions mentioned herein may be applied to that sequence stretch in the CH3 region.
  • a homodimerization promoter is located in a CH4-CH4 assembly interface (e.g. in case of IgM or IgE). Accordingly, the homodimerization promoter may be located in a CH4 domain, preferably in the region or the amino acid sequence that generates/defmes a CH4-CH4 interface between two antibody heavy chains.
  • the homodimerization promoter comprises at least one amino acid substitution in an amino acid sequence contributing to a CH3-CH3 assembly interface. In preferred embodiments, the homodimerization promoter comprises at least two amino acid substitutions in an amino acid sequence contributing to a CH3-CH3 assembly interface.
  • the at least one amino acid substitution may preferably located in aa345-aa441 (numbering according to EU numbering of the CH3 domain) or an analogous region of a CH3 domain.
  • an “analogous region” in different types of heavy chains e.g. antibodies of a different species, isotype, subclass and/or allotype.
  • the homodimerization promoter comprises at least one amino acid substitution in an amino acid sequence contributing to a CH4-CH4 assembly interface (e.g. in case of IgM or IgE). In preferred embodiments, the homodimerization promoter comprises at least two amino acid substitution in an amino acid sequence contributing to a CH4-CH4 assembly interface.
  • the at least one amino acid substitution preferably at least two amino acid substitutions, are located in the contact residues contributing to a CH3-CH3 assembly interface selected from amino acid positions 345, 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 360, 364, 366, 368, 370, 390, 392, 394, 395, 397, 399, 400, 405, 407, 409, 411 , 439, 441 (numbering according to EU numbering of the CH3 domain) or analogous positions of a CH3 domain.
  • the at least one amino acid substitution preferably at least two amino acid substitutions, are located in the contact residues contributing to a CH3-CH3 assembly interface selected from amino acid positions 345, 347, 349, 350, 351 , 354, 355, 356, 357, 360, 364, 366, 368, 370, 392, 394, 399, 405, 407, 409 (numbering according to EU numbering of the CH3 domain) or analogous positions of a CH3 domain.
  • the skilled person will be able to identify an “analogous positions” in different types of heavy chains (e.g. antibodies of a different species, isotype, subclass and/or allotype).
  • the at least one homodimerization promoter comprises at least one amino acid substitution that destroys or destabilizes the naturally occurring CH3-CH3 interface of an antibody heavy chain, thereby preventing assembly of the HC or HC element to a non-modified or to a wild-type antibody heavy chain.
  • the homodimerization promoter as defined herein comprises at least one assembly element.
  • the term “assembly element” relates to an element that can be comprised in a homodimerization promoter of the invention.
  • An “assembly element” comprises at least one, preferably at least two amino acid substitutions that are positioned in an antibody heavy chain or HC element.
  • a homodimerization promoter comprises only one “assembly element” (e.g. as further described herein for homodimerization promoter Homo12) or may comprise up to three “assembly elements” (e.g. as further described herein for homodimerization promoter Homo04), or even more than three “assembly elements”.
  • the assembly elements as further defined herein are particularly suitable for IgG 1 derived heavy chains or HC elements (given amino acid substitutions numbering according to EU numbering of the CH3 domain) or lgG3 derived heavy chains or HC elements (given amino acid substitutions with reference to SEQ ID NO: 249, P01860-1).
  • an “assembly element” is defined herein through amino acid substitutions with reference to lgG1 , the skilled person will be able to introduce “analogous amino acid substitutions” into different types of heavy chains (e.g. lgG3 or antibodies of a different species, isotype, subclass and/or allotype).
  • the homodimerization promoter comprises or consists of at least one selected from a steric steering assembly element, electrostatic steering assembly element, interchain disulfide assembly element, or any combination thereof.
  • the respective elements comprise at least one amino acid substitution located/positioned in a CH3-CH3 or CH4-CH4 assembly interface.
  • a given assembly element as defined herein may sometimes act via a combination of different steering mechanisms, e.g. steric steering and electrostatic steering.
  • An example in this context is the assembly element 10 (D399R and K409D), which may act through electrostatic steering, but also steric steering.
  • electrostatic steering element
  • this assembly element is referred to as electrostatic steering (element)
  • electrostatic steering element
  • some steric steering assembly elements i.e. mainly acting via steric steering
  • the homodimerization promoter comprises or consists of at least one steric steering assembly element.
  • a steric assembly element sterically promotes, supports, forces, or directs the pairing or the assembly of two identical antibody heavy chains or HC elements, wherein the antibody heavy chains or HC elements are provided by the nucleic acid sequence.
  • a steric assembly element sterically prevents or reduces the pairing or the assembly to e.g. wildtype or reference antibody heavy chains or non-modified antibody heavy chains.
  • the at least one steric steering assembly element comprises at least one knob-modification and/or at least one hole-modification. In preferred embodiments, the at least one steric steering assembly element comprises at least one knob-modification and at least one hole-modification.
  • knock modification has to be understood as a moiety, e.g. an amino acid substitution, wherein an amino acid with a small (or smaller) side chain volume (e.g. A, S, T, L, V etc.) is substituted with an amino acid with a larger side chain volume to generate a “knob”.
  • a homodimerization promoter of the invention may comprise at least one knob modification.
  • said amino acid residue having a larger side chain volume is selected from the group consisting of e.g. arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
  • R, F, Y, or W may be introduced (preferably by substituting another amino acid residue) to generate a “knob” or protuberance in the at least one antibody heavy chain or HC element.
  • hole modification has to be understood as an amino acid substitution, wherein an amino acid with a large (or larger) side chain volume (e.g. R, F, Y, W, L etc.) is substituted with an amino acid with a smaller side chain volume to generate a “hole”.
  • a “hole” has to be understood as a cavity in at least one antibody heavy chain or HC element (provided by the nucleic acid sequence) that is suitable for sterically interacting with a compatible “knob” modification or protuberance on a corresponding antibody heavy chain or HC element (provided by the nucleic sequence).
  • a homodimerization promoter of the invention may comprise at least one hole modification.
  • an amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), Leucine (L), valine (V).
  • A, S, T, L, or V may be introduced (preferably by substituting another amino acid residue) to generate a “hole” or cavity in at least one antibody heavy chain or HC element.
  • the at least one knob-modification and at least one hole-modification both comprise at least one amino acid substitution. In preferred embodiments, the at least one knob-modification and at least one holemodification both comprise at least one amino acid substitution in a CH3-CH3 or CH4-CH4 assembly interface.
  • the homodimerization promoter comprises or consists of at least one electrostatic steering assembly element.
  • the at least one electrostatic steering assembly element comprises at least one positivecharge-modification and/or at least one negative-charge-modification.
  • “Positive-charge modification” in this context means substituting at least one neutral or negatively charged amino acid with a positively charged amino acid, preferably Arginine, Lysine, Histidine, more preferably Arginine or Lysine.
  • “Negative-charge modification” in this context means substituting at least one neutral or positively charged amino acid with a positively charged amino acid (Aspartic acid, Glutamic acid).
  • the at least one positive-charge-modification and at least one negative-charge-modification both comprise at least one amino acid substitution introducing a positive and negative charge, respectively.
  • the at least one amino acid substitutions are D399R and K409D (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions.
  • the skilled person will of course be able to apply these specified amino acid substitutions also to lgG3 heavy chains (D329R and K339D; numbering according to the amino acid positions of SEQ ID NO: 249, P01860-1) or antibodies of a different species, isotype, subclass and/or allotype.
  • the homodimerization promoter comprises or consists of at least one interchain disulfide assembly element.
  • an interchain disulfide assembly element is at least one amino acid residue, suitably a Cysteine residue, that is integrated into the at least one antibody heavy chain or HC element amino acid sequence to allow the formation of disulphide bonds.
  • the antibody heavy chain or HC element comprises at least one amino acid substitution, preferably a Cysteine substitution, to allow specific assembly and covalent connection (via C-C bonds) of the two antibody heavy chains or HC elements to promote the formation of a homodimeric HC.
  • the at least one interchain disulfide assembly element comprises at least one, preferably a least two disulfide modifications.
  • the at least one interchain disulfide assembly element comprising at least one or two of the following amino acid substitutions: S364C, F405C, L368C, Y349C, Y407C, K370C, D399C, L365C, K409C, T366C, L406C, T411C, L351C, P353C, S408C, V369C, V363C, E357C, L398C, P395C, K392C, N390C, T394C, Q347C, P352C, T393C, K439C, D356C, Q362C, S400C, K360C, S354C.
  • the at least two disulfide modifications are amino acid substitutions introducing a Cysteine, preferably Y349C and S354C (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions.
  • a Cysteine preferably Y349C and S354C (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions.
  • the skilled person will of course be able to apply these specified amino acid substitutions also to lgG3 heavy chains (Y279C and S284C with reference to SEQ ID NO: 249, P01860-1 ) or antibodies of a different species, isotype, subclass and/or allotype.
  • the homodimerization promoter comprises at least one of the following assembly elements comprising the specified amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions:
  • - assembly element 6 Y407V, T366Y, L368A;
  • - assembly element 10 D399R, K409D (electrostatic steering assembly element);
  • - assembly element 11 Y349C, S354C (interchain disulfide assembly element); - assembly element 12: D399M, K409V;
  • - assembly element 16 Y349T, S364H;
  • - assembly element 17 Y349D, T366K;
  • - assembly element 18 T366M, S364Q, K370Y, E357D, Y349S, E356G, Y407A, K409V;
  • - assembly element 22 Y349D, R355E, T366K.
  • the homodimerization promoter comprises at least one of the following assembly elements selected from assembly element 1 , 4, 5, 6, 7, 8, 10, 11.
  • the homodimerization promoter comprises at least one of the following assembly elements comprising the specified amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions:
  • - assembly element 10 D399R, K409D (electrostatic steering assembly element).
  • the homodimerization promoter comprises or consists of at least two assembly elements as defined herein. In preferred embodiments, the homodimerization promoter comprises or consists of at least two assembly elements selected from assembly element 1 to 22 (as defined herein), preferably selected from assembly element 1 , 4, 5, 6, 7, 8, 10, 11.
  • the homodimerization promoter comprises or consists of at least two assembly elements (AEs) selected from
  • - assembly element 10 D399R, K409D (electrostatic steering assembly element).
  • D399R, K409D electrostatic steering assembly element.
  • the skilled person will of course be able to apply these specified amino acid substitutions also to lgG3 heavy chains with reference to SEQ ID NO: 249 (P01860-1) or antibodies of a different species, isotype, subclass and/or allotype.
  • the homodimerization promoter comprises or consists of assembly element 1 and assembly element 4. In embodiments the homodimerization promoter comprises or consists of assembly element 1 and assembly element 5. In embodiments the homodimerization promoter comprises or consists of assembly element 1 and assembly element 8. In embodiments the homodimerization promoter comprises or consists of assembly element 1 and assembly element 10. In embodiments the homodimerization promoter comprises or consists of assembly element 4 and assembly element 5. In embodiments the homodimerization promoter comprises or consists of assembly element 4 and assembly element 8. In embodiments the homodimerization promoter comprises or consists of assembly element 4 and assembly element 10. In embodiments the homodimerization promoter comprises or consists of assembly element 5 and assembly element 8. In embodiments the homodimerization promoter comprises or consists of assembly element 5 and assembly element 10. In embodiments the homodimerization promoter comprises or consists of assembly element 8 and assembly element 10.
  • the homodimerization promoter additionally comprises at least one interchain disulfide assembly element as defined herein, preferably Y349C and S354C, and at least one or two steric steering assembly elements as defined herein, preferably selected from assembly element 1 , 4, 5, 8, or 10.
  • the homodimerization promoter additionally comprises at least one electrostatic steering assembly element as defined herein, preferably D399R and K409D, and at least one or two steric steering assembly elements as defined herein, preferably selected from assembly element 1 , 4, 5, or 8.
  • the homodimerization promoter comprises assembly element 1 (E345R, Q347R, K360E; or analogous amino acid substitutions) and/or assembly element 11 (Y349C, S354C; or analogous amino acid substitutions) and, optionally, at least one further assembly element as defined herein.
  • assembly elements as defined herein preferably assembly elements 1 to 22 as defined herein, are combined and included into homodimerization promoters of the invention.
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter comprising the specified amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions selected from:
  • E345R, Q347R, K360E, K392L, T394W, F405A (comprising AEs 1 and 4);
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter located in a CH3 domain from IgG 1 , the homodimerization promoter comprising the specified amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions selected from:
  • Homo02 Y349C, S354C, K392L, T394W, F405A, (comprising AEs 11 and 4);
  • Homo05 E345R, Q347R, Y349C, S354C, K360E, (comprising assembly element 1 and 11);
  • Homo06 T350V, L351 Y, T366L, K392L, T394W, F405A, Y407V, (comprising AEs 8 and 5);
  • Homo08 E345R, Q347R, K360D, T366V, D399M, Y407A, K409V, (comprising AEs 3 and 9);
  • Homo09 D399R, K409D, (comprising AE 10);
  • Homol 0 Y349C, S354C, T366Y, L368A, Y407V, (comprising AEs 11 and 6);
  • Homol 1 Y349S, E356G, E357D, S364Q, T366M, K370Y, Y407A, K409V, (comprising AE 18);
  • Homol 2 F405L, K409R, (comprising AE 21);
  • Homol 3 Y349D, R355E, T366K, (comprising AE 22).
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter selected from Homo01 - Homol 0.
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter, preferably located in a CH3 domain from lgG1 , comprising the specified amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions selected from:
  • the homodimerization promoters as defined herein, in particular Homo01 to Homo13 do not assemble to a different type of homodimerization promoter.
  • an antibody heavy chain or HC element comprising Homo01 does not assemble to an antibody heavy chain or HC element comprising Homo02 to Homo13, in particular does not assemble to an antibody heavy chain Homo03, Homo06, and Homo09.
  • the homodimerization promoters as defined herein, in particular Homo01 to Homol 3 do not assemble to wild type (or non-modified reference) antibody heavy chain or HC elements.
  • the at least one antibody heavy chain or HC element comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 96-108, 110-122, 124-136, or a fragment or variant of any of these, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein (Homo01 to Homol 3) are comprised in the respective sequences:
  • the at least one antibody heavy chain or HC element comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 138-150, 152-164, 166-178, 180-192, 194-206, 208-220, or a fragment or variant of any of these, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined (Homo01 to Homol 3) are comprised in the respective sequences:
  • Homo04 SEQ ID NOs: 141, 155, 169, 183, 197, 211;
  • Homo05 SEQ ID NOs: 142, 156, 170, 184, 198, 212;
  • Table 1 summarizes the SEQ ID NOs for three different lgG1 sections (aa345-aa409; hinge-CH2-CH3; CH1-hinge- CH2-CH3) and the 3 different allotypes G1m3,1 (R120, D12/L14; referred to as R-DEL), G1m3,-1 (R120, E12/M14; referred to as R-EEM) and G1m17,1 (K120, D12/L14; referred to as K-DEL).
  • Table 1 Overview of lqG1 amino acid sequences (SEQ ID NOs) comprising homodimerization promoters
  • homodimerization promoter for lgG3 are provided. These homodimerization promoter are composed of assembly elements as defined herein (e.g. assembly element 1 to 22) that were adapted to the respective lgG3 heavy chains, e.g. the position with reference to lgG3 has been adapted with reference to SEQ ID NO: 249 (P01860-1).
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter located in a CH3 domain from lgG3, the homodimerization promoter comprising the specified amino acid substitutions (numbering according to the amino acid positions of SEQ ID NO: 249 (P01860-1 )) or analogous amino acid substitutions selected from:
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter selected from Homo01 ’ - Homol O’.
  • the at least one antibody heavy chain or HC element comprises a homodimerization promoter comprising the specified amino acid substitutions (numbering according to the amino acid positions of SEQ ID NO: 249 (P01860-1)) or analogous amino acid substitutions selected from:
  • the at least one antibody heavy chain or HC element comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 222-234, or a fragment or variant of any of these, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein (Homo01 ’-13’) are comprised in the respective sequences:
  • the at least one antibody heavy chain or HC element comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 236-248, 250-262, or a fragment or variant of any of these, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein (Homo01 ’-13’) are comprised in the respective sequences:
  • Table 2 summarizes the SEQ ID NOs for three different lgG3 sections (aa275-aa339; hinge-CH2-CH3; CH1 -hinge- CH2-CH3).
  • Table 2 Overview oflqG3 amino acid sequences (SEQ ID NOs) comprising homodimerization promoters
  • two produced antibody heavy chain or HC element comprising polypeptides interact via the homodimerization promoter in a parallel C2-symmetry to form homodimers.
  • the nucleic acid upon administration of the nucleic acid to a cell or subject, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the produced antibody heavy chains or HC elements form homodimers. In equally preferred embodiments, upon administration of the nucleic acid to a cell or subject, less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% of the produced antibody HC elements are non-assembled HC elements. Mass spectrometry (MS) or non-reducing Western blot analzyes can be used to determine the percentage of assembled HC homodimers and/or detect non-assembled antibody chains (half-bodies).
  • MS mass spectrometry
  • Western blot analzyes can be used to determine the percentage of assembled HC homodimers and/or detect non-assembled antibody chains (half-bodies).
  • the produced antibody upon administration of the nucleic acid to a cell or subject, retains a functionality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% compared to a corresponding antibody lacking a homodimerization promoter.
  • the functionality of the produced antibody upon administration of the nucleic acid to a cell or subject, is not reduced by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% compared to a corresponding antibody lacking a homodimerization promoter.
  • the functionality of an antibody can be e.g. determined using an ELISA assay, for example by using ELISAs determining antigen or Fc receptor binding.
  • the produced antibody upon administration of the nucleic acid to a cell or subject, retains a (production) yield or antibody titer of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% compared to a corresponding antibody lacking a homodimerization promoter.
  • the nucleic acid additionally encodes for at least one antibody light chain (LC) or LC element that is preferably derived or selected from an antibody LC, or a fragment or variant thereof.
  • LC antibody light chain
  • LC element relates to a polypeptide element selected or derived from an antibody light chain. Such an “antibody LC element” may be a fragment of an antibody light chain or may be a full antibody LC.
  • the at least one antibody light chain or LC element is derived or selected from an antibody as defined herein, preferably depending on the antibody heavy chain or HC element that is encoded by the nucleic acid.
  • the at least one antibody light chain or LC element is derived or selected from a K or X LC, or a fragment or variant of any of these, preferably derived or selected from a human K LC, or a fragment or variant thereof.
  • the at least one LC element is N-terminally or C-terminally fused to the at least one HC element. In preferred embodiments, the at least one LC element is N-terminally or C-terminally fused to the variable region the HC element. In preferred embodiments, the at least one LC element is N-terminally fused to HC element, preferably fused to the variable region of the HC element. In preferred embodiments, the at least one LC element is C-terminally fused to HC element, preferably fused to the variable region of HC element.
  • the at least one antibody light chain or LC element is a variable region of an antibody LC, or a variant thereof, preferably of a human K LC, or a variant thereof.
  • the at least one coding sequence encoding the at least one antibody heavy chain or HC element additionally encodes at least one antibody light chain or LC element.
  • Having the light-chain, in particular the variable domain of a light chain, fused to a heavy chain has the advantage that by introducing homodimerization promoters as defined herein, functional assembled antibodies can be generated via one single nucleic acid species. This also reduces the complexity of e.g. compositions expressing multiple different antibodies, because for each correctly assembled antibody only one nucleic acid construct is needed.
  • the antibody light chain or LC element is fused to the antibody heavy chain or HC element, preferably via a linker peptide element, e.g. a flexible linker peptide element such as a Glycine-Serine linker.
  • a linker peptide element e.g. a flexible linker peptide element such as a Glycine-Serine linker.
  • the antibody light chain or LC element is fused to a variable region of an antibody HC or variant, preferably via a linker peptide element.
  • the antibody LC element is a full-length LC and fused to an antibody HC comprising a VH, CH1 , CH2, CH3 domain and hinge region, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: VL-CL-[preferab/y linker]-VH-CH1-hinge-CH2-CH3 (encoding for monomer of scFab-IgG).
  • the antibody LC element is a VL and fused to an antibody HC variant comprising a VH, CH2, CH3 domain and hinge region, but not a CH1 domain, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: l -preferably linker]-VH-hinge-CH2-CH3 (encoding for monomer of scFv- Fc with VL-VH orientation).
  • the antibody LC element is a VL and interspersed between the VH and CH2 domain of an antibody HC variant comprising a VH, CH2, CH3 domain and hinge region, but not a CH1 domain, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: W- ⁇ preferably linker]-VL-hinge-CH2- CH3 (encoding for monomer of scFv-Fc with VH-VL orientation).
  • the at least one coding sequence additionally encodes at least one 2A peptide, that are preferably characterized by a polypeptide sequence selected from SEQ ID NOs: 1434-1508 of WO2017/081082, or fragments or variants of these sequences, and SEQ ID NOs: 264 or 265, or fragments or variants of these sequences, and, optionally an additional peptide comprising a furin cleavage site upstream of the 2A peptide.
  • a suitable furin cleavage site may be selected from a sequence according to SEQ ID NO: 263.
  • the at least one coding sequence comprises at least one nucleic acid sequence encoding an antibody heavy chain or HC element and at least one nucleic acid sequence encoding an antibody light chain or LC element that are separated by a nucleic acid sequence encoding for the 2A peptide and optionally the furin cleavage site upstream of the 2A peptide.
  • the at least one antibody light chain or LC element is encoded by an additional coding sequence, preferably wherein the nucleic acid is bicistronic.
  • Embodiments where the light chain is provided via a separate coding sequence may require the introduction of further antibody chain assembly promoters to facilitate correct HC-LC assembly (e.g. HC-LC assembly promoter, LC-HC assembly promoter).
  • further antibody chain assembly promoters to facilitate correct HC-LC assembly (e.g. HC-LC assembly promoter, LC-HC assembly promoter).
  • the at least one antibody heavy chain or HC element comprises a heavy chain - light chain (HC-LC) assembly promoter.
  • HC-LC heavy chain - light chain
  • HC-LC assembly promoter relates to a moiety (e.g. an amino acid) that promotes, supports, forces, or directs assembly of at least one antibody heavy chain and at least one antibody light chain.
  • a moiety e.g. an amino acid
  • such a moiety is typically at least one amino acid substitution capable of promoting, supporting, forcing, or directing a certain assembly of the at least two antibody polypeptide chains.
  • an amino acid substitution is a substitution that does not occur naturally, suitably, a substitution that does not occur naturally in human antibody chains.
  • an “HC-LC assembly promoter” may be located on an antibody heavy chain to promote, support, force, or direct an assembly between the two antibody chains (HC and LC).
  • an HC-LC antibody chain assembly promoter promotes, supports, forces, or directs assembly of at least two antibody polypeptide chains (HC and LC) preferably in the presence of an additional antibody polypeptide chain (or additional polypeptide chains).
  • suitable HC-LC assembly promoters may promote, support, force, or direct (correct) assembly of at least two antibody polypeptide chains (HC and LC) while, at the same time, avoiding assembly to other antibody polypeptide chains lacking an antibody chain assembly promoter, comprising a different HC-LC antibody chain assembly promoter or comprising a non-corresponding LC-HC antibody chain assembly promoter (see below).
  • HCs and LCs are co-translationally translocated into the ER of a B-cell, and folding begins before the polypeptide chains are completely translated.
  • Most IgGs assemble first as HO dimers to which LCs are added covalently via a disulphide bond between the CL and CH1 domains.
  • a typical antibody heavy chain comprises a natural antibody heavy chain - light chain assembly sequence interface, forming a CH1-CL interface that mediates assembly. It has to be emphasized that such naturally occurring antibody heavy chain - light chain assembly interfaces are not comprised by the term “HC-LC assembly promoter” as used herein.
  • a HC-LC assembly promoter as defined herein is located on a heavy chain or HC element and specifically interacts with a LC-HC assembly promoter on a light chain or LC element (as further specified below) to promote specific assembly of LCs to HCs.
  • the at least one HC-LC assembly promoter is located in the constant region of the antibody heavy chain or HC element. In preferred embodiments in that context, the at least one HC-LC assembly promoter is located in the Fab region of the antibody heavy chain or HC element. In preferred embodiments, the at least one HC- LC assembly promoter is located in the CH1 domain region of the antibody heavy chain or HC element. In preferred embodiments in that context, the at least one HC-LC assembly promoter comprises at least one amino acid substitution in an amino acid sequence of the HC-LC assembly interface. In particular, the at least one HC-LC assembly promoter comprises at least one amino acid substitution in an amino acid sequence of the CH1-CL interface.
  • the at least one HC-LC assembly promoter comprises or consists of at least one selected from steric steering assembly element, electrostatic steering assembly element, SEED assembly element, DEKK assembly element, interchain disulfide assembly element, or any combination thereof.
  • the at least one HC-LC assembly promoter comprises at least one steric assembly element, wherein the steric assembly element comprises a modification selected from at least one knob-modification and/or at least one hole modification.
  • the at least one light chain or LC element comprises a light chain - heavy chain (LC-HC) assembly promoter.
  • LC-HC light chain - heavy chain assembly promoter
  • a moiety e.g. an amino acid
  • such a moiety is typically at least one amino acid substitution capable of promoting, supporting, forcing, or directing a certain assembly of the at least two antibody polypeptide chains.
  • an amino acid substitution is a substitution that does not occur naturally, suitably, a substitution that does not occur naturally in human antibody chains.
  • an “LC-HC assembly promoter” may be located on an antibody light chain to promote, support, force, or direct an assembly between the two antibody chains (HC and LC).
  • an LC-HC antibody chain assembly promoter promotes, supports, forces, or directs assembly of at least two antibody polypeptide chains (LC and HC) preferably in the presence of an additional antibody polypeptide chain (or additional polypeptide chains).
  • suitable LC-HC assembly promoters may promote, support, force, or direct (correct) assembly of at least two antibody polypeptide chains (HC and LC) while, at the same time, avoiding assembly to other antibody polypeptide chains lacking an antibody chain assembly promoter, comprising a different LC-HC antibody chain assembly promoter, or comprising a noncorresponding HC-LC antibody chain assembly promoter (see above).
  • LCs and HCs are co-translationally translocated into the ER of a B-cell, and folding begins before the polypeptide chains are completely translated.
  • Most IgGs assemble first as HC dimers to which LCs are added covalently via a disulphide bond between the CL and CH1 domains.
  • a typical antibody light chain comprises a natural antibody light chain - heavy chain assembly sequence interface, forming a CL-CH1 interface that mediates assembly. It has to be emphasized that such naturally occurring antibody light chain - heavy chain assembly interfaces are not comprised by the term “LC-HC assembly promoter" as used herein.
  • the at least one LC-HC assembly promoter is located in the constant region of the antibody light chain or LC element. According to preferred embodiments, the at least one LC-HC assembly promoter is located in the Fab region of the antibody light chain or LC element. According to preferred embodiments, the at least one LC-HC assembly promoter is located in the CL domain of the antibody light chain or LC element. According to preferred embodiments, the at least one LC-HC assembly promoter comprises at least one amino acid substitution in an amino acid sequence of the LC-HC assembly interface.
  • the at least one LC-HC assembly promoter comprises or consists of at least one selected from steric steering assembly element, electrostatic steering assembly element, SEED assembly element, DEKK assembly element, interchain disulfide assembly element, or any combination thereof.
  • the HC-LC assembly promoter is located in a constant and/or variable region of the at least one antibody heavy chain or HC element.
  • the HC- LC assembly promoter is located in a CH1 and/or VH domain of the at least one antibody heavy chain or HC element.
  • the at least one antibody light chain or LC element comprises a light chain - heavy chain (LC-HC) assembly promoter.
  • the LC-HC assembly promoter is located in a constant and/or variable region of the at least one antibody heavy chain or LC element.
  • the LC-HC assembly promoter is located in a CL and/or VL domain of the at least one antibody heavy chain or LC element.
  • the HC-LC and/or the LC-HC assembly promoter comprise at least one amino acid residue that does not occur naturally at the respective position or at least one amino acid sequence that does not occur naturally at the respective position.
  • the HC-LC and/or the LC- HC assembly promoter comprise at least one amino acid substitution in an amino acid sequence contributing to a HC- LC assembly interface.
  • the HC-LC and LC-HC assembly promoter are corresponding HC-LC and LC-HC assembly promoters selected from the CrossMab CH1 - CL , orthogonal Fab VHVRDICH1CRD2 - VL RDICACRD2, orthogonal Fab VHvRD2CH1wt - VLwozCAwt, TCR CaC(3, CR3, MUT4, DuetMab technology (Brinkmann & Kontermann (2017; mAbs; PMID 28071970; e.g. Table 2).
  • the corresponding HC-LC and LC-HC assembly promoters jointly support, promote, force, or direct the assembly of the at least one antibody heavy chain or HC element and the at least one antibody light chain or LC element.
  • the HC-LC and LC-HC assembly promoter prevent or reduce assembly of the antibody HC and LC (or element) with LC and HC (or elements) that do not comprise the corresponding HC-LC and LC-HC assembly promoter, respectively, or no such promoter.
  • the nucleic acid encodes at least one functional antibody (comprising correctly assembled heavy and light chains (or elements), or more than one functional antibody (e.g. 2, 3, 4, or more antibodies)
  • nucleic acid of the invention e.g. type of nucleic acid, structure of nucleic acid, elements of nucleic acid, modification of nucleic acid etc.
  • said features defining nucleic acid features or sequences may also apply to the nucleic acid in any other aspect of the invention (e.g. nucleic acid set, composition, combination, kit, medical uses).
  • the nucleic acid is a monocistronic, a bicistronic, or a multicistronic nucleic acid. In particularly preferred embodiments, the nucleic acid is a monocistronic nucleic acid.
  • the nucleic acid is an artificial nucleic acid as defined herein.
  • the nucleic acid is monocistronic and the coding sequence of said nucleic acid encodes at least two different peptides or proteins. Accordingly, said coding sequence may encode at least two, three, four, five, six, seven, eight and more antibody chains as defined herein, linked with or without an amino acid linker sequence, wherein said linker sequence can comprise rigid linkers, flexible linkers, cleavable linkers, or a combination thereof.
  • a monocistronic nucleic acid may comprise a coding sequence encoding a HC or HC element linked with or without an amino acid linker sequence to (at least a fragment of) LC.
  • the nucleic acid may be bicistronic or multicistronic and comprises at least two coding sequences. Said at least two coding sequences suitably encode two or more different antibody chains as specified herein. Accordingly, the coding sequences in a bicistronic or multicistronic nucleic acid suitably encodes distinct proteins or peptides as defined herein or fragments variants thereof. Preferably, the coding sequences in said bicistronic or multicistronic constructs may be separated by at least one IRES (internal ribosomal entry site) sequence.
  • IRES internal ribosomal entry site
  • a bicistronic nucleic acid may comprise a nucleic acid sequence encoding at least a HC or HC element and a nucleic acid sequence encoding an LC or LC element, wherein, optionally, the respective coding sequences are separated by at least one IRES.
  • suitable IRES sequences may be selected from the list of nucleic acid sequences according to SEQ ID NOs: 1566-1662 of the patent application W02017081082, or fragments or variants of these sequences. In this context, the disclosure of WO2017081082 relating to IRES sequences is herewith incorporated by reference.
  • the nucleic acid e.g. the DNA or RNA
  • the nucleic acid is a modified and/or stabilized nucleic acid, preferably a modified and/or stabilized artificial nucleic acid.
  • the nucleic acid may thus be provided as a “stabilized artificial nucleic acid” or “stabilized coding nucleic acid” that is to say a nucleic acid showing improved resistance to in vivo degradation and/or a nucleic acid showing improved stability in vivo, and/or a nucleic acid showing improved translatability in vivo.
  • specific suitable modifications/adaptations in this context are described which are suitable to “stabilize” the nucleic acid.
  • the nucleic acid sequences of the present invention may be provided as a “stabilized RNA”, “stabilized coding RNA”, “stabilized DNA” or “stabilized coding DNA”.
  • the nucleic acid has a half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 day or at least 14 days (e.g. upon in vivo administration of the composition).
  • the nucleic acid comprises at least one coding sequence encoding at least one antibody heavy chain or HC element that comprises a homodimerization promoter as defined herein.
  • the nucleic acid comprises or consists of at least one coding sequence encoding at least one antibody heavy chain or HC element that comprises a homodimerization promoter as defined herein, preferably encoding any one SEQ ID NOs: 96-108, 110-122, 124-136, 138-150, 152-164, 166-178, 180-192, 194-206, 208-220, 222-234, 236-248, 250-262, or fragments of variants thereof.
  • the at least one coding sequence of the nucleic acid is a codon modified coding sequence.
  • the amino acid sequence encoded by the at least one codon modified coding sequence is not being modified compared to the amino acid sequence encoded by the corresponding wild type or reference coding sequence.
  • the at least one coding sequence of the nucleic acid is a codon modified coding sequence, wherein the codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.
  • the at least one codon modified coding sequence is a G/C optimized coding sequence.
  • the at least one coding sequence of the artificial nucleic acid preferably the RNA
  • the at least one coding sequence of the nucleic acid has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
  • the nucleic acid comprising the codon modified coding sequence When transfected into mammalian host cells, the nucleic acid comprising the codon modified coding sequence has a stability of between 12-18 hours, orgreaterthan 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and is capable of being expressed by the mammalian host cell.
  • the nucleic acid comprising the codon modified coding sequence is translated into protein, wherein the amount of protein is at least comparable to, or preferably at least 10% more than, or at least 20% more than, or at least 30% more than, or at least 40% more than, or at least 50% more than, or at least 100% more than, or at least 200% or more than the amount of protein obtained by a naturally occurring or wild type or reference coding sequence transfected into mammalian host cells.
  • the nucleic acid may be modified, wherein the C content of the at least one coding sequence may be increased, preferably maximized, compared to the C content of the corresponding wild type or reference coding sequence (herein referred to as “C maximized coding sequence”).
  • C maximized coding sequence The generation of a C maximized nucleic acid sequences may suitably be carried out using a modification method according to WO2015062738. In this context, the disclosure of WO2015062738 is included herewith by reference.
  • the nucleic acid may be modified, wherein the G/C content of the at least one coding sequence may be optimized compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as “G/C optimized coding sequence”).
  • G/C optimized coding sequence refers to a coding sequence wherein the G/C content is preferably increased to the essentially highest possible G/C content.
  • the generation of a G/C content optimized nucleic acid sequence may be carried out using a method according to W02002098443. In this context, the disclosure of W02002098443 is included in its full scope in the present invention.
  • the nucleic acid may be modified, wherein the codons in the at least one coding sequence may be adapted to human codon usage (“human codon usage adapted coding sequence”). Codons encoding the same amino acid occur at different frequencies in humans. Accordingly, the coding sequence of the nucleic acid is preferably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage.
  • the nucleic acid may be modified, wherein the G/C content of the at least one coding sequence may be modified compared to the G/C content of the corresponding wild type or reference coding sequence (“G/C modified coding sequence”).
  • G/C optimization’ or “G/C content modification” relate to a nucleic acid that comprises a modified, preferably an increased number of G and/or C nucleotides as compared to the corresponding wild type or reference coding sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides.
  • the nucleic acid may be modified, wherein the codon adaptation index (CAI) may be increased or preferably maximised in the at least one coding sequence (“CAI maximized coding sequence”). It is preferred that all codons of the wild type or reference nucleic acid sequence that are relatively rare in e.g. a human are exchanged for a respective codon that is frequent in the e.g. a human, wherein the frequent codon encodes the same amino acid as the relatively rare codon. Suitably, the most frequent codons are used for each amino acid of the encoded protein (see Table 2 of published PCT patent application WO2021156267, most frequent human codons are marked with asterisks).
  • the RNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95, most preferably 1 (CA ).
  • the at least one coding sequence comprises a nucleic acid sequence (encoding an antibody HC element comprising a homodimerization promoter) wherein the nucleic acid sequence encodes an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 96-108, 110-122, 124-136, 138-150, 152-164, 166-178, 180-192, 194-206, 208-220, 222-234, 236-248, 250-262, or a fragment or variant of any of these, wherein the specified or analogous
  • the nucleic acid encodes an amino acid sequence that is identical or at least is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 138, 152, 166, 180, 194, 208, 140, 154, 168, 182, 196, 210, 143, 157, 171, 185, 199, 213, 146, 160, 174, 188, 202, 216, 236, 250, 238, 252, 240, 254, 241, 255, 244, 258, or a fragment or variant of any of these, wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter Homo01 , Homo03, Homo06, Homo09, Homo01’, Homo03’, Homo06’, Homo09’, Homo01, Ho
  • the at least one coding sequence comprises more than one stop codon to allow sufficient termination of translation. In particularly embodiments, the at least one coding sequence comprises two or three stop codon to allow sufficient termination of translation, optionally positioned in alternating reading frames.
  • the nucleic acid comprises a protein-coding region (“coding sequence” or“cds”), and a 5’- UTR and/or 3’-UTR.
  • UTRs may harbour regulatory sequence elements that determine nucleic acid, preferably RNA turnover, stability, and localization.
  • UTRs may harbour sequence elements that enhance translation.
  • translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy.
  • Certain combinations of 3’-UTRs and/or 5’-UTRs may enhance the expression of operably linked coding sequences encoding peptides or proteins as defined herein. Nucleic acid molecules harbouring said UTR combinations enable rapid and transient expression of encoded proteins after administration.
  • the nucleic acid comprises at least one untranslated region (UTR).
  • the nucleic acid comprises a 3’ UTR and a 5’ UTR.
  • the at least one UTR is selected from at least one heterologous 3-UTR.
  • the nucleic acid comprises at least one 3-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
  • the 3-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.
  • the nucleic acid comprises at least one 3-UTR, wherein the at least one 3-UTR comprises or consists of a nucleic acid sequence derived or selected from a 3-UTR of a gene selected from PSMB3, ALB7, alphaglobin, beta-globin, ANXA4, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1, RPS9, SLC7A3, TUBB4B, or from a homolog, a fragment, or variant of any of these.
  • the at least one heterologous 3’-UTR comprises or consists of a nucleic acid sequence derived from a 3’-UTR of a gene selected from PSMB3, ALB7, alpha-globin, beta-globin, ANXA4, CASP1 , COX6B1 , FIG4, GNAS, NDUFA1 , RPS9, SLC7A3 or TUBB4B, or from a homolog, a fragment or variant of any one of these genes, preferably wherein the at least one heterologous 3’-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 48-89, or a fragment or variant of any of these.
  • the artificial nucleic acid comprises a 3’-UTR derived or selected from a PSMB3 gene.
  • the at least one heterologous 3’-UTR (derived from PSMB3) comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 48, 49, 78-89, or a fragment or variant thereof, preferably being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 49, or a fragment or variant thereof
  • the nucleic acid comprises a 3’-UTR as described in WO2016107877, the disclosure of WO2016107877 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WQ2016107877, or fragments or variants of these sequences.
  • the artificial nucleic acid comprises a 3’-UTR as described in WQ2017036580, the disclosure of WQ2017036580 relating to 3’-UTR sequences herewith incorporated by reference. Suitable 3’-UTRs are SEQ ID NOs: 152-204 of WO2017036580, or fragments or variants of these sequences.
  • the artificial nucleic acid comprises a 3-UTR as described in WO2016022914, the disclosure of WO2016022914 relating to 3’- UTR sequences herewith incorporated by reference.
  • Preferred 3’-UTRs are nucleic acid sequences according to SEQ ID NOs: 20-36 of WO2016022914, or fragments or variants of these sequences.
  • the at least one UTR is selected from at least one heterologous 5-UTR.
  • the nucleic acid comprises at least one 5-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
  • the 5’-UTR comprises one or more of a binding site for proteins that affect a nucleic acid stability or nucleic acid location in a cell, or one or more miRNA or binding sites for miRNAs. Accordingly, miRNA or binding sites for miRNAs as defined above may be removed from the 5-UTR or introduced into the 5-UTR in order to tailor the expression of the nucleic acid to desired cell types or tissues.
  • the nucleic acid comprises at least one 5-UTR, wherein the at least one 5-UTR comprises a nucleic acid sequence derived or selected from a 5’-UTR of gene selected from HSD17B4, RPL32, AIG1 , alphaglobin, ASAH1 , ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL35A, SLC7A3, TUBB4B, UBQLN2, or from a homolog, a fragment or variant of any one of these genes,
  • the at least one heterologous 5-UTR comprises or consists of a nucleic acid sequence derived from a 5-UTR of a gene selected from HSD17B4, RPL32, AIG1 , alpha-globin, ASAH1 , ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL35A, SLC7A3, TUBB4B, UBQLN2, or a homolog, a fragment or variant of any one of these genes, preferably wherein the at least one heterologous 3-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12-47, or a fragment or variant of any of these.
  • the nucleic acid comprises a 5’-UTR derived or selected from a HSD17B4 gene.
  • the at least one heterologous 5’-UTR (derived from HSD17B4) comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12, 13, 46, 47, or a fragment or variant thereof, preferably being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13, or a fragment or variant thereof.
  • the nucleic acid comprises a 5’-UTR as described in WO2013143700, the disclosure of WO2013143700 relating to 5-UTR sequences herewith incorporated by reference.
  • Particularly preferred 5’-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 ofW02013143700, or fragments or variants of these sequences.
  • the nucleic acid comprises a 5’-UTR as described in WO2016107877, the disclosure of WO2016107877 relating to 5'-UTR sequences herewith incorporated by reference.
  • Particularly preferred 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 of WO2016107877, or fragments or variants of these sequences.
  • the nucleic acid comprises a 5-UTR as described in WO2017036580, the disclosure of
  • WO2017036580 relating to 5-UTR sequences herewith incorporated by reference.
  • Preferred 5’-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 of WO2017036580, or fragments or variants of these sequences.
  • the nucleic acid comprises a 5’-UTR as described in WO2016022914, the disclosure of
  • WO2016022914 relating to 5-UTR sequences herewith incorporated by reference.
  • Preferred 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 of WO2016022914, or fragments or variants of these sequences.
  • the nucleic acid preferably the RNA, comprises at least one coding sequence as specified herein, operably linked to a 3-UTR and/or a 5-UTR selected from the following 5’-UTR/3’-UTR combinations: a-1 (HSD17B4/PSMB3), a-2 (NDUFA4/PSMB3), a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1), b-5 (NOSIP/COX6B1), c-1 (NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4 (NDUFA4 /NDUFA1), c-5 (ATP5A1/PS
  • the at least one heterologous 5-UTR is selected from HSD17B4 and the at least one heterologous 3’ UTR is selected from PSMB3 (a-1 (HSD17B4/PSMB3)).
  • the artificial nucleic acid comprises a ribosome binding site, also referred to as “Kozak sequence” identical to or at least 80%, 85%, 90%, 95% identical to SEQ ID NOs: 1 , 2, or sequences GCCGCCACC (DNA), GCCGCCACC (RNA), GCCACC (DNA), GCCACC (RNA), ACC (DNA) or ACC (RNA), or fragments or variants thereof.
  • the “Kozak sequence” comprises or consists of RNA sequence ACC.
  • the nucleic acid comprises at least one poly(N) sequence, e.g. at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or combinations thereof.
  • the nucleic acid e.g. the RNA, comprises at least one poly(A) sequence as defined herein.
  • the artificial nucleic acid comprises least two, three, or more poly(A) sequences.
  • the at least one poly(A) sequence preferably wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosines, more preferably about 60 to about 250 adenosines, most preferably about 60 to about 150 adenosines.
  • the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides, preferably consecutive adenosine nucleotides.
  • the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100).
  • the at least one poly(A) sequence comprises about 100 consecutive adenosine nucleotides.
  • the nucleic acid comprises at least one interrupted poly(A) sequence.
  • the nucleic acid may comprise at least one interrupted poly(A) sequence preferably comprising about 100 adenosine nucleotides or more, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, preferably by at least about 10 non-adenosine (N10) nucleotides.
  • N10 non-adenosine
  • the poly(A) sequence as defined herein may be located directly at the 3' terminus of the nucleic acid, preferably the RNA.
  • the 3’-terminal nucleotide (that is the last 3’-terminal nucleotide in the polynucleotide chain) is the 3’-terminal A nucleotide of the at least one poly(A) sequence.
  • the term “directly located at the 3’ terminus” has to be understood as being located exactly at the 3’ terminus - in other words, the 3’ terminus of the nucleic acid consists of a poly(A) sequence terminating with an A. Ending on an adenosine nucleotide decreases the induction of interferons, e.g.
  • the at least one poly(A) sequence is located at the 3’ terminus, optionally, wherein the 3’ terminal nucleotide is an adenosine.
  • the nucleic acid e.g. the RNA
  • the nucleic acid comprises at least one poly(A) sequence obtained by enzymatic polyadenylation, wherein the majority of RNA molecules comprise about 100 (+/- 20) to about 500 (+/- 100) adenosine nucleotides, preferably about 100 (+/- 20) to about 200 (+/- 40) adenosine nucleotides.
  • the nucleic acid comprises at least one polyadenylation signal.
  • the nucleic acid of the invention comprises at least one poly(C) sequence and/or at least one miRNA binding site and/or at least one histone stem-loop sequence.
  • the nucleic acid e.g. the RNA, comprises at least one histone stem-loop (hSL) or histone stem-loop structure.
  • hSL in the context of the invention may be located in an UTR region, preferably in the 3’ UTR.
  • the term “histone stem-loop” (hSL) is intended to refer to nucleic acid sequences that forms a stem-loop secondary structure predominantly found in histone mRNAs.
  • Histone stem-loop sequences/structures may suitably be selected from hSL sequences as disclosed in WO2012019780, the disclosure relating to histone stem-loop sequences/histone stem-loop structures incorporated herewith by reference.
  • a hSL sequence that may be used within the present invention may be derived from formulae (I) or (II) of WO2012019780.
  • the artificial nucleic acid e.g. the RNA, comprises at least one hSL sequence derived from at least one of the specific formulae (la) or (Ila) of W02012019780.
  • the artificial nucleic acid comprises a 3’-terminal sequence element.
  • the 3’-terminal sequence element represents the 3’ terminus of the RNA.
  • a 3’-terminal sequence element may comprise at least one poly(N) sequence as defined herein and, optionally, at least one hSL as defined herein.
  • the nucleic acid comprises at least one 3’-terminal sequence element comprising or consisting of an RNA sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-11 , or a fragment or variant of these sequences.
  • the nucleic acid comprises a 3’- terminal sequence element comprising a hSL as defined herein followed by a poly(A) sequence comprising about 100 consecutive adenosines.
  • the nucleic acid comprises a 3’-terminal sequence element comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or 6, or a fragment or variant thereof.
  • the nucleic acid comprises a 5’-terminal sequence element comprising or consisting of a nucleic acid sequence, preferably an RNA sequence, being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequences AGGAGA, GGGAGA, GGGAAA, AGAAUA, AGAUUA, GAUGGG or GGGCG, or a fragment or variant of these sequences.
  • the nucleic acid comprises a 5’-terminal sequence element comprising or consisting of a nucleic acid sequence, preferably an RNA sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence AGGAGA, or a fragment or variant thereof.
  • a 5’-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase.
  • the first nucleotide of said 5’-terminal start sequence may preferably comprise a 2’0 methylation, e.g. 2’0 methylated guanosine or a 2’0 methylated adenosine.
  • the nucleic acid is an isolated nucleic acid.
  • isolated nucleic acid does not comprise a cell or a subject that comprises said nucleic acid, but relates to the artificial nucleic acid as an isolated molecule or ensemble of isolated molecules.
  • the “isolated nucleic acid” can be an artificial nucleic acid isolated or purified from a cell, or can be an artificial nucleic acid (e.g. RNA) isolated from an RNA in vitro transcription.
  • the “isolated nucleic acid” has been produced synthetically (e.g. via in vitro methods) and purified.
  • the nucleic acid of the invention is selected from a DNA.
  • the DNA may be any type of DNA that comprises a coding sequence as defined herein including any type of single stranded DNA, any type of double stranded DNA, any type of linear DNA, and any type of circular DNA.
  • the nucleic acid is an RNA.
  • the RNA may be any type of RNA that comprises a coding sequence as defined herein including any type of single stranded RNA, any type of double stranded RNA, any type of linear RNA, and any type of circular RNA.
  • the RNA is selected from mRNA, circular RNA, replicon RNA, or viral RNA.
  • the RNA is a circular RNA.
  • “circular RNA” or “circRNAs” have to be understood as an RNA construct that is connected to form a circle and therefore does not comprise a 3' or 5’ terminus.
  • said circRNA comprises at least one coding sequence as defined herein.
  • RNA is a replicon RNA.
  • the term “replicon RNA” or “self-replicating RNA” will be recognized and understood by the person of ordinary skill in the art and is preferably intended to be an optimized self-replicating RNA.
  • Such constructs may include replicase elements derived from e.g. alphaviruses (e.g. SFV, SIN, VEE, or RRV) and the substitution of the structural virus proteins with the nucleic acid of interest.
  • the RNA is selected from an mRNA.
  • the nucleic acid preferably the RNA of the invention comprises at least one modified nucleotide.
  • the at least one modified nucleotide is selected from pseudouridine, N1- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2- thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydrops
  • the nucleic acid preferably the RNA, comprises at least one modified nucleotide selected from pseudouridine (i ) or N1 -methylpseudouridine (ml qj).
  • the nucleic acid, preferably the RNA comprises at least one modified nucleotide selected from N1- methylpseudouridine (m1ip).
  • essentially all, e.g. essentially 100% of the uracil in the coding sequence (or the full nucleic acid sequence) have a chemical modification, preferably a chemical modification in the 5-position of the uracil.
  • 100% of the uracil in the full nucleic acid sequence, preferably the RNA sequence are substituted with N1 -methylpseudouridine (ml ip).
  • 100% of the uracil in the full nucleic acid sequence, preferably the RNA sequence are substituted with pseudouridine (ip).
  • Incorporating modified nucleotides such as e.g. pseudouridine (ip) or N1 -methylpseudouridine (ml ip) into the coding sequence (or the full nucleic acid sequence) may be advantageous as unwanted innate immune responses (upon administration of the RNA) may be adjusted or reduced (if required).
  • modified nucleotides such as e.g. pseudouridine (ip) or N1 -methylpseudouridine (ml ip) into the coding sequence (or the full nucleic acid sequence) may be advantageous as unwanted innate immune responses (upon administration of the RNA) may be adjusted or reduced (if required).
  • the nucleic acid preferably the RNA, does not comprise chemically modified nucleotides.
  • the nucleic acid, preferably the RNA of the invention does not comprise N1- methylpseudouridine (m1"+’) substituted positions or pseudouridine (ip) substituted positions.
  • m1"+’ N1- methylpseudouridine
  • ip pseudouridine
  • a 5’-cap structure as defined below is typically not considered to be a chemically modified nucleotide.
  • the nucleic acid, preferably the RNA comprises a sequence that consists only of G, C, A and U nucleotides and therefore does not comprise modified nucleotides, and optionally comprises a 5’-cap structure.
  • the nucleic acid preferably the RNA
  • a 5’-cap structure suitably stabilizes the nucleic acid and/or enhances expression of the encoded protein and/or reduces the stimulation of the innate immune system after administration.
  • the 5’-cap structure is selected from a m7G, capO, cap1 , cap2, modified capO or a modified cap1 structure.
  • a 5'-cap (capO or cap1) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co-transcriptional capping) using cap analogues.
  • cap analogue as used herein will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of an RNA molecule when incorporated at the 5’-end of the nucleic acid molecule.
  • Non-polymerizable means that the cap analogue will be incorporated only at the 5’- terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3’-direction by a templatedependent polymerase, particularly, by template-dependent RNA polymerase.
  • the “cap analogue” is formed by using a tri-nucleotide cap analogue as disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, WO2017066797, and W02023007019.
  • the nucleic acid preferably the RNA comprises a cap1 or a modified cap1.
  • the cap1 structure is formed via co-transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG.
  • a particularly preferred cap1 analog in that context is m7G(5’)ppp(5’)(2’OMeA)pG.
  • the cap1 structure is a modified cap1 structure and is formed using co-transcriptional capping using tri-nucleotide cap analogue 3'OMe-m7G(5 , )ppp(5')(2'OI ⁇ /leA)pG.
  • the 5’-cap structure is formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases) to generate capO or cap1 or cap2 structures.
  • capping enzymes e.g. vaccinia virus capping enzymes and/or cap-dependent 2’-0 methyltransferases
  • the nucleic acid is preferably an RNA that provides at least one coding sequence as defined herein encoding at least one antibody HO element that comprises a homodimerization promoter as defined herein that is produced after administration to a cell or subject.
  • the RNA of the invention may comprises a 5' Cap structure as defined herein, a 5’ UTR as defined herein, a 3’ UTR as defined herein, hSL as defined herein, Poly(A)sequence as defined herein, and optional chemical modifications as defined herein.
  • the RNA is preferably an in vitro transcribed RNA (e.g. an in vitro transcribed mRNA).
  • the RNA is lyophilized (e.g. according to WO2016165831 or WO2011069586) to yield a temperature stable dried RNA.
  • the RNA may also be dried using spray-drying or spray-freeze drying (e.g. according to WO2016184575 or WO2016184576) to yield a temperature stable RNA (powder).
  • the RNA of the invention is a GMP-grade RNA, particularly a GMP-grade mRNA.
  • GMP- grade RNA is produced using a manufacturing process approved by regulatory authorities.
  • RNA production is performed under current good manufacturing practice (GMP), implementing quality control steps on DNA and RNA level, preferably quality controls selected from methods described in WO2016180430.
  • GMP current good manufacturing practice
  • the nucleic acid is a purified RNA.
  • the RNA has been purified by at least one step of purification.
  • the term “purified RNA” has to be understood as RNA which has a higher purity after certain purification steps than the starting material.
  • Typical impurities that are essentially not present in purified RNA comprise peptides or proteins (e.g. enzymes derived from DNA dependent RNA in vitro transcription, e.g.
  • RNA polymerases RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, RNA fragments (short double stranded RNA fragments, abortive sequences etc.), free nucleotides (modified nucleotides, NTPs, cap analogue), template DNA, buffer components (HEPES, TRIS, MgCI2) etc.
  • Other potential impurities may be derived from e.g. fermentation procedures (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents etc.).
  • the nucleic acid is a purified RNA, preferably wherein the RNA has been purified by RP- HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
  • the RNA has been purified using RP-HPLC (preferably as described in W02008077592) and/or TFF (preferably as described in WO2016193206) and/or oligo d(T) purification (preferably as described in WO2016180430) to e.g. to remove dsRNA, non-capped RNA and/or RNA fragments.
  • the RNA has been purified by a step of 5’ dephosphorylation of linear RNA, DNA digestion, protein digestion, and/or dsRNA digestion.
  • the purified RNA has a purity level of at least about 70%, 75%, 80%, 85%, 90%, or 95%, preferably more than 95%.
  • the degree of purity is determined by an analytical HPLC method.
  • the nucleic acid preferably the RNA of the invention has a certain integrity.
  • integration generally describes whether the complete nucleic acid sequence or RNA sequence is present. Low RNA integrity could be due to, amongst others, RNA degradation, RNA cleavage, incorrect or incomplete chemical synthesis of the RNA, incorrect base pairing, integration of modified nucleotides or the modification of already integrated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete RNA in vitro transcription.
  • the nucleic acid preferably the RNA has an integrity of at least about 50%, preferably of at least about 60%, more preferably of at least about 70%, most preferably of at least about 80% or about 90% or higher. Integrity is suitably determined using analytical HPLC, preferably analytical RP-HPLC.
  • the nucleic acid is a therapeutic nucleic acid and suitable for use in treatment or prevention of a disease, disorder or condition. Accordingly, the nucleic acid is suitably used in a therapeutic context, in particular to provide a therapeutic antibody.
  • the nucleic acid comprises the following elements:
  • the nucleic acid preferably the mRNA, comprises the following elements:
  • a 5-UTR preferably selected or derived from a 5-UTR of a HSD17B4 gene
  • a 3'-UTR preferably selected or derived from a 3-UTR of a PSMB3 gene
  • the nucleic acid is an mRNA that comprises in the following order: A) a cap1 structure as defined herein; B) a 5-UTR derived from a HSD17B4 gene as defined herein;
  • G optionally, chemically modified nucleotides, suitably selected from i or ml i , wherein ml ip is preferred.
  • the present invention relates inter alia to a nucleic acid set that encodes an antibody, or a fragment of an antibody, or a variant of an antibody.
  • nucleic acid of the first aspect may be applied to the nucleic acid set of the second aspect.
  • features and embodiments described in the context of the nucleic acid set of the second aspect may likewise be applied to the nucleic acid of the first aspect, the antibody of the third aspect, the composition of the fourth aspect, the combination of the fifth aspect, and any further aspect of the invention.
  • nucleic acid set preferably means a combined occurrence of different nucleic acids (e.g. a first nucleic acid providing a HC or HC element and a second nucleic acid providing an LC or LC element). “Combined occurrence” means that the individual components of the nucleic acid set may be provided as (physically) separate entities or as a combined entity or any combination thereof.
  • a “nucleic acid set” comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids.
  • a nucleic acid set preferably encodes for all HC and LC (or elements) of an antibody, or more than one antibody (e.g. 2, 3, 4, 5, or more).
  • the nucleic acid set comprises a first nucleic acid selected from a nucleic acid as defined in the first aspect preferably comprising at least one coding sequence encoding at least one antibody heavy chain or HC element that comprises a homodimerization promoter as defined herein, and a second or additional nucleic acid comprising at least one coding sequence encoding at least one antibody light chain or LC element.
  • the antibody (that is encoded by the nucleic acid set) is derived or selected from an antibody that is characterized by any of the features as defined in the context of the first aspect.
  • the antibody is derived or selected from a full-length antibody, preferably derived or selected from a human lgG1 or lgG3, in particular from a full-length human lgG1 or lgG3.
  • the antibody light chain or LC element that is provided by the second nucleic acid is derived or selected from a K or A LC, or a fragment or variant of any of these, preferably derived or selected from a human K LC, or a fragment or variant thereof.
  • the antibody heavy chain or HC element that is provided by the first nucleic acid comprises a HC-LC assembly promoter as defined herein.
  • the antibody light chain or LC element that is provided by the second nucleic acid comprises a LC-HC assembly promoter as defined herein.
  • the second nucleic acid is characterized by any of the generic features provided in section “Nucleic acid features and embodiments" of the first aspect.
  • the second nucleic acid is an RNA, preferably an mRNA.
  • all nucleic acid constructs of the set are selected from RNA, preferably mRNA.
  • the nucleic acid set of the second aspect is an RNA set, preferably an mRNA set.
  • the second nucleic acid comprises the following elements:
  • the second nucleic acid preferably the RNA, comprises the following sequence elements:
  • a 5’-UTR preferably selected or derived from a 5-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3-UTR of a PSMB3 gene
  • the second nucleic acid is an mRNA that comprises the following elements in 5’- to 3’-direction:
  • G optionally, chemically modified nucleotides, suitably selected from ip or ml i , wherein ml ip is preferred.
  • the nucleic acid set of the present aspect comprises a) a first nucleic acid comprising at least one cds encoding at least one heavy chain or HC element as defined herein and at least one homodimerization promoter; b) a second nucleic acid comprising at least one cds encoding at least one light chain or LC element as defined herein.
  • the nucleic acid set of the present aspect comprises a) a first nucleic acid comprising at least one coding sequence encoding at least one heavy chain or HC element as defined herein and at least one homodimerization promoter and at least one HC-LC assembly promoter; b) a second nucleic acid comprising at least one coding sequence encoding at least one light chain or LC element as defined herein and at least one LC-HC assembly promoter.
  • the heavy chain or HC element encoding nucleic acid and the light chain or LC element encoding nucleic acid are comprised in the composition in a w/w ratio ranging between about 10:1 to 1:10 (e.g., between 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1 :6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1 :3, or 2:1 to 1 :2).
  • the heavy chain or HC element encoding nucleic acid and the light chain or LC element encoding nucleic acid are comprised in the composition in a molar ratio ranging between approximately 10:1 to 1:10 (e.g., between 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2).
  • the produced antibodies upon administration of the nucleic acid set to a cell or subject, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the produced antibodies are correctly assembled, preferably comprised of a HC (or HC element) homodimer and two assembled LCs (or LC elements).
  • the produced antibody upon administration of the nucleic acid set to a cell or subject, retains a (production) yield or antibody titer of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% compared to a corresponding antibody lacking a homodimerization promoter.
  • the invention relates inter alia to an antibody or a fragment of an antibody, or a variant of an antibody.
  • nucleic acid of the first aspect or the nucleic acid set of the second aspect may likewise be applied to the antibody of the third aspect.
  • features and embodiments described in the context of the antibody of the third aspect may likewise be applied to any further aspect of the invention.
  • the antibody is encoded by any nucleic acid as defined in the context of the first aspect or by any of the nucleic acid sets of the second aspect.
  • the antibody is derived or selected from an antibody that is characterized by any of the features as defined in the context of the first and second aspect.
  • the antibody is derived or selected from a full-length antibody, preferably derived or selected from a human lgG1 or lgG3.
  • the antibody HC or HC element of the antibody comprises at least one amino acid sequence that comprises amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions as defined in the context of the first aspect, preferably selected from assembly element 1 to assembly element 22.
  • the antibody HC or HC element of the antibody comprises at least one amino acid sequence that comprises amino acid substitutions (numbering according to EU numbering of the CH3 domain) or analogous amino acid substitutions selected from assembly element 1 , assembly element 4, assembly element 5, assembly element 8, and assembly element 10.
  • the antibody heavy chain or HC element of the antibody comprises at least one amino acid sequence that comprises at least one homodimerization promoter sequence selected from Homo01 to Homo13.
  • the antibody heavy chain or HC element of the antibody preferably IgG 1
  • the antibody HC or HC element of the antibody comprises an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 96-108, 110-122, 124-136, SEQ ID NOs: 138- 150, 152-164, 166-178, 180-192, 194-206, 208-220, or a fragment or variant thereof, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein are comprised.
  • the antibody HC or HC element of the antibody comprises an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 138-150, 152-164, 166-178, ISO- 192, 194-206, 208-220, or a fragment or variant thereof, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein are comprised.
  • the antibody heavy chain or HC element of the antibody comprises a homodimerization promoter located in a CH3 domain from lgG3, the homodimerization promoter comprising the specified amino acid substitutions (numbering according to the amino acid positions of SEQ ID NO: 249, P01860-1) or analogous amino acid substitutions selected from Homo01’ to Homo13’.
  • the antibody HC or HC element of the antibody comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 222-234, 236-248, 250-262, or a fragment or variant thereof, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein are comprised.
  • the antibody HC or HC element of the antibody comprises at least one amino acid sequence comprising or consisting of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 236-248, 250-262, or a fragment or variant thereof, preferably wherein the specified or analogous amino acid substitutions of the respective homodimerization promoter as defined herein are comprised.
  • the antibody additionally comprises at least one light chain or LC element.
  • the light chain or LC element is fused to the antibody heavy chain or HC element of the antibody, preferably via a linker peptide element.
  • the antibody light chain or LC element is fused to a variable region of an antibody HC or variant, preferably via a linker peptide element.
  • the antibody LC element is a full-length LC and fused to an antibody HC comprising a VH, CH1 , CH2, CH3 domain and hinge region, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: VL-CL-[preferably linker]-VH-CH1 -hinge-CH2-CH3 (encoding for monomer of scFab-IgG).
  • the antibody LC element is a VL and fused to an antibody HC variant comprising a VH, CH2, CH3 domain and hinge region, but not a CH1 domain, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: VL-[preferably linker]-VH-hinge-CH2-CH3 (encoding for monomer of scFv-Fc with VL-VH orientation).
  • the antibody LC element is a VL and interspersed between the VH and CH2 domain of an antibody HC variant comprising a VH, CH2, CH3 domain and hinge region, but not a CH1 domain, preferably via a linker peptide element, resulting in following sequence from N- to C-terminus: VH-[preferably linker]-VL-hinge-CH2-CH3 (encoding for monomer of scFv-Fc with VH-VL orientation).
  • the antibody comprises at least one LC or LC element as a separate polypeptide chain.
  • the antibody light chain or LC element can comprise at least one LC-HC assembly promoter and the antibody heavy chain or HC element can comprise at least one HC-LC assembly promoter.
  • the present invention relates to a composition forexpression of one antibody, preferably for expression of one or more than one or a plurality of different antibodies in a cell or a subject.
  • nucleic acid of the first aspect may likewise be applied to the composition of the present aspect.
  • nucleic acid set of the second aspect may likewise be applied to any further aspect of the invention.
  • the composition comprises at least one nucleic acid as defined in the context of the first aspect and/or at least one nucleic acid set as defined in the context of the second aspect. In preferred embodiments, the composition comprises at least two nucleic acids of the first aspect and/or at least two nucleic acid sets of the second aspect.
  • introducing homodimerization promoters into heavy chains or HC elements as defined herein allows for the provision of nucleic acid compositions that encode more than one antibody as the likelihood of HC-HC mismatch is strongly reduced or prevented.
  • the composition is for expression of at least two different antibodies in a cell.
  • the composition is for expression of at least two (correctly) assembled antibodies in the same cell.
  • the composition is for expression of at least two different antibodies in vivo, e.g. in a subject, preferably a human subject.
  • said composition is for expression of at least two (correctly) assembled antibodies in vivo, e.g. in a subject, preferably a human subject.
  • the composition encodes at least two different antibodies, preferably a plurality of different antibodies, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100.
  • the composition encodes 2-100, 2-50, 2-25, 2-10, preferably 2 to 5 different antibodies.
  • each of the different antibodies may specifically recognize and/or bind to at least one different target as defined herein.
  • said at least one different target is a different epitope or different antigen as defined herein.
  • the composition encodes for at least two, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 antibodies, wherein each antibody comprises a different homodimerization promoter.
  • the homodimerization promoters as defined herein prevent or reduce misassembly with HCs or HC elements comprising a different homodimerization promoter or lacking a homodimerization promoter.
  • the homodimerization promoters differ in at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more amino acid positions, preferably in at least 3-12 amino acid positions, with regard to every other homodimerization promoter.
  • the difference in amino acid positions between two given homodimerization promoters is found in the specified or analogous amino substitutions of the two given homodimerization promoters.
  • the following particular preferred homodimerization promoters differ as follows:
  • the homodimerization promoters differ in at least one assembly element with regard to every other homodimerization promoter.
  • the homodimerization promoters of the antibodies of the composition are selected such that the assembly of homodimeric antibody HC elements comprising a respective homodimerization promoter is characterized by a more favorable (lower) binding energy than the assembly of the respective HC element comprising a respective homodimerization promoter to a different HC element comprising a different homodimerization promoter.
  • the homodimerizat'ion promoters of the antibodies of the composition are selected such that an assembly to different homodimerization promoters of the antibodies of the composition is prevented (e.g., a HC or HC element comprising Homo01 does not bind to Homo03, Homo05, Homo06 or Homo09; a HC or HC element comprising Homo03 does not bind to Homo01 , Homo05, Homo06 or Homo09; etc.)
  • the at least one nucleic acid and/or nucleic acid set of the composition encodes for 2, 3, 4, 5, 6, 7, 8, 9, 10 antibodies, wherein each of the antibodies comprises a different homodimerization promoter selected from Homo01 to Homol 3 and/or HomoOT to Homo13’, preferably Homo01/03/06/09 and/or Homo01703705706709’ .
  • the at least one nucleic acid and/or at least one nucleic acid set of the composition encodes for 2, 3, 4 antibodies, wherein each of the antibodies comprises a different homodimerization promoter selected from:
  • the at least one nucleic acid and/or at least one nucleic acid set of the composition encode for 2, 3, 4 antibodies, wherein each of the antibodies comprises a different homodimerization promoter selected from:
  • the composition comprises at least one additional nucleic acid and/or nucleic acid set encoding for at least one antibody that does not comprise a homodimerization promoter (in particular, comprising a non-modified (wildtype) heavy chain or HC element).
  • the composition comprises at least one additional nucleic acid or nucleic acid set encoding for an antibody comprising a non-modified (wildtype) HC assembly interface.
  • the composition comprises at least one additional nucleic acid and/or nucleic acid set encoding for at least one antibody comprising heterodimeric antibody HC elements and comprising a heterodimerization promoter pair.
  • Suitable heterodimerization promoter pairs may comprise HC-HC assembly promoter pairs HC-HC-PP1 to HC-HC-PP18, preferably HC-HC-PP3 to HC-HC-PP5, or HC-HC-PP18 as described in WO2022023559.
  • a different promoter pair per encoded antibody is used.
  • the at least one encoded antibody of the composition is derived or selected from a, preferably human, IgG 1 , lgG3 or a fragment or variant of any of these. Suitable antibodies are described in the context of the first and second aspect. In preferred embodiments, at least one, optionally all antibodies are selected or derived from a scFv-Fc and/or scFab-IgG, preferably a scFv-Fc.
  • the composition comprises at least one additional nucleic acid encoding for a single-chain antibody.
  • the composition comprises at least one additional nucleic acid (or set) encoding for a nonmodified (wildtype) antibody, e.g. an antibody comprising a non-modified (wildtype) HC assembly interface.
  • a nonmodified (wildtype) antibody e.g. an antibody comprising a non-modified (wildtype) HC assembly interface.
  • the at least one non-modified (wildtype) antibody or a fragment or variant thereof comprises a heavy chain of an antibody or a fragment or variant thereof, and/or a light chain of an antibody or a fragment or variant thereof that does not comprise an antibody assembly promoter preferably as described in the context of the invention.
  • the term “does not comprise an antibody assembly promoter” as described in the context of the invention” has not to be understood as a light chain and/or heavy chain that is lacking (naturally occurring) assembly interfaces.
  • the heavy chain of such an antibody or a fragment or variant thereof, and/or a light chain of such an antibody may comprise antibody chain assembly interfaces.
  • said (naturally occurring) assembly interfaces do not assemble with any one of the antibody chain assembly promoters as described in the context of the invention (in particular, do not assemble with the homodimerization promoters).
  • the at least one additional antibody or antibody fragment or antibody variant (that does not comprise an assembly promoter as defined herein) is derived or selected from a monoclonal antibody or fragments thereof, a chimeric antibody or fragments thereof, a human antibody or fragments thereof, a humanized antibody or fragments thereof, an intrabody or fragments thereof, or a single chain antibody or fragments thereof, or a nanobody or fragments thereof.
  • the at least one additional antibody or antibody fragment or variant thereof is derived or selected from lgG1, lgG2, lgG3, lgG4, IgD, lgA1, lgA2, IgE, IgM, IgNAR, hcIgG, BiTE, diabody, DART, TandAb, scDiabody, sc- Diabody-CH3, Diabody-CH3, Triple Body, mini antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv- CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Fab, Fab 1 , Fc, Facb, pFc'
  • the composition comprises 2 to 100 different nucleic acids or sets encoding at least one antibody or a fragment or variant thereof (as defined herein). In preferred embodiments, the composition of the invention comprises 2 to 20 different nucleic acids or sets encoding at least one antibody or a fragment or variant thereof (as defined herein). In more preferred embodiments, the composition of the invention comprises 2 to 10 or 2 to 5 different nucleic acids or sets encoding at least one antibody or a fragment or variant thereof (as defined herein).
  • composition encodes for:
  • n antibodies comprising a homodimerization promoter, wherein n is an integer of 1 , 2, 3, 4;
  • - o antibodies comprising a non-modified (wildtype) HC assembly interface, wherein o is an integer of 0, 1;
  • - p antibodies comprising a heterodimerization promoter pair, wherein p is an integer of 0, 1 , 2, 3;
  • the n antibodies are provided by at least one nucleic acid or nucleic acid set as defined herein comprising at least one homodimerization promoter as defined herein, preferably wherein the n antibodies comprise a different homodimerization promoter selected from Homo01 - Homol 3 and/or Homo01 ’- Homol 3', preferably Homo01 , Homo03, Homo06, or Homo09.
  • the o antibodies are provided by at least one nucleic acid or nucleic acid set as defined comprising non-modified or wildtype heavy chains or HC elements (e.g. encoding for an lgG1 or lgG3 with non-modified heavy chains or HC elements).
  • the p antibodies are provided by at least one nucleic acid or nucleic acid set comprising at least one heterodimerization promoter pair as defined herein, preferably wherein the p antibodies comprise a different heterodimerization promoter pair selected from HC-HC-PP1 to HC-HCPP18, preferably HC-HC- PP3, HC-HC-PP4, HC-HC-PP5, or HC-HC-PP18 as described in WO2022023559.
  • the q single-chain antibodies are provided by at least one nucleic acid and comprise single-chain antibodies (e.g. BiTE) which intrinsically do not need guided HC-HC assembly or HC-LC assembly.
  • n is an integer of 1
  • 2, 3, o is an integer of 1.
  • the n antibodies comprise different homodimerization promoters, preferably selected from Homo01, Homo03, Homo06, Homo09.
  • the n antibodies are HC-LC fusion constructs as defined herein.
  • the o antibody may an IgG 1 or lgG3 with non-modified heavy chains, optionally a respective HC-LC fusion construct.
  • p is an integer of 1 , 2, or 3 and the p antibodies comprise different heterodimerization promoters as defined herein.
  • q is an integer of 1 , 2, or 3.
  • administration of the composition to a cell or to a subject leads to expression of at least two assembled antibodies (or fragments or variants) in said cell or subject, optionally to expression of 2 to 40, preferably 2,
  • administration of the composition to a cell or to a subject leads to expression of at least two assembled antibodies (or fragments or variants), optionally to expression of 2 to 40, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • administration of the composition to a cell or to a subject leads to expression of at least two assembled antibodies (or fragments or variants) in the presence of at least one different antibody chain (a non-modified (wildtype) antibody) wherein, preferably, more than about 50%, 60%, 70%, 75%, 80%, 90%, 95%, preferably more than about 90% of the produced antibodies are correctly assembled.
  • administration of the composition to a cell or to a subject leads to expression of at least one assembled antibody (or fragment or variant) in the presence of at least one different antibody chain (a non-modified (wildtype) antibody), wherein, preferably, less than about 50%, 40%, 30%, 20%, 10%, 5%, preferably less than about 10% of the produced antibodies are misassembled and/or non-assembled antibody chains (half-bodies).
  • the subject is a human subject.
  • Mass spectrometry (MS) or non-reducing Western blot analyses can be used to determine the percentage of assembled/misassembled antibodies as well as non-assembled antibody chains (half-bodies).
  • administration of the composition to a cell or subject leads to expression of at least two assembled antibodies (or fragments or variants), optionally to expression of 2 to 40, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the composition is for in vivo expression of two different correctly assembled antibodies. In more preferred embodiments, the composition is for in vivo expression of three different correctly assembled antibodies. In even more preferred embodiments, the composition is for in vivo expression of four different correctly assembled antibodies. In particularly preferred embodiments, the composition is for in vivo expression of five different correctly assembled antibodies
  • the nucleic acid constructs of the composition are characterized by any of the generic features provided in section “Nucleic acid features and embodiments” of the first aspect. Accordingly, the nucleic acid constructs of the composition are preferably RNA, more preferably mRNA constructs. Preferably, the composition is an mRNA composition. Preferred features of RNA constructs are described in “Nucleic acid features and embodiments" of the first aspect, in particular section “Preferred nucleic acid constructs”.
  • the composition comprises at least one pharmaceutically acceptable carrier or excipient.
  • the at least one pharmaceutically acceptable carrier or excipient of the composition may preferably be selected to be suitable for systemic or local administration to a subject, preferably a human subject.
  • the composition is a pharmaceutical composition, preferably a sterile and/or pyrogen-free pharmaceutical composition.
  • the nucleic acids and/or nucleic acid sets are complexed or associated with at least one further compound to obtain a formulated composition.
  • a formulation may have the function of a transfection agent.
  • a formulation may also protect the nucleic acid (e.g. the RNA) from degradation.
  • the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) encoding different antibodies are co-formulated. Accordingly, the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) are formulated (complexed/associated) as one entity. In these embodiments, the formulation/complexation of the nucleic acids and/or nucleic acid sets is the same. In other embodiments, the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) encoding different antibodies are formulated separately, preferably wherein one entity comprises a nucleic acid or nucleic acid set encoding for one (complete) antibody.
  • nucleic acids and/or nucleic acid sets are formulated (complexed/ associated) as separate entities.
  • some nucleic acids and/or nucleic acid sets are co-formulated, and some nucleic acids and/or nucleic acid sets are formulated separately.
  • those separate formulations may be administered in a spatially separated and/or timely staggered manner, e.g. to further reduce antibody misassembly.
  • nucleic acids and/or nucleic acid sets e.g.
  • RNA or RNA sets encoding for one (complete) antibody are co-formulated to increase the probability that all nucleic acid sequences are present in one particle/formulation to ensure that the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) are up taken by the same cell (upon administration).
  • a co-formulation of the nucleic acids and/or nucleic acid sets may be advantageous for the production of correctly assembled antibodies (upon administration to a cell).
  • the nucleic acids and/or nucleic acid sets are complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound.
  • Complexation/association (“formulation”) to cationic or polycationic compounds as defined herein facilitates the uptake of the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) of the composition into cells.
  • the one or more cationic or polycationic compound is selected from a cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.
  • the at least one cationic or polycationic compound is selected from a cationic or polycationic peptide or protein.
  • Preferred cationic or polycationic proteins or peptides that may be used for complexation of the nucleic acid or set can be derived from formula (Arg)l;(Lys)m;(His)n;(Om)o;(Xaa)x of the patent application W02009030481 orWO2011026641, the disclosure of W02009030481 or WO2011026641 relating thereto incorporated herewith by reference.
  • the nucleic acids and/or nucleic acid sets e.g.
  • RNA or RNA sets is complexed, or at least partially complexed, with at least one cationic or polycationic proteins or peptides preferably selected from an amino acid sequence identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 90-94, or any combinations thereof.
  • nucleic acids and/or nucleic acid sets e.g. RNA or RNA sets
  • RNA or RNA sets is complexed or associated with one or more lipids, thereby forming lipid-based carriers.
  • lipid-based carriers encompasses lipid-based delivery systems for nucleic acid (e.g. RNA) that comprise a lipid component.
  • a lipid-based carrier may additionally comprise other components suitable for encapsulating/incorporating/complexing a nucleic acid including a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
  • a typical “lipid-based carrier” is selected from liposomes, lipid nanoparticles (LNPs), lipoplexes, solid lipid nanoparticles, and/or nanoliposomes.
  • nucleic acids and/or nucleic acid sets may completely or partially incorporated or encapsulated in a lipid-based carrier, wherein the nucleic acid may be located in the interior space of the lipid-based carrier, within the lipid layer/membrane of the lipid-based carrier, or associated with the exterior surface of the lipid-based carrier.
  • the incorporation of nucleic acid, preferably the RNA, into lipid-based carriers may be referred to as "encapsulation".
  • a “lipid-based carrier” is not restricted to any particular morphology, and can include any morphology generated when at least one lipid is combined in an aqueous environment in the presence of nucleic acid (e.g. RNA).
  • Lipid-based carriers can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50nm and 500nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposomes a specific type of lipid-based carrier, are characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • the nucleic acid e.g. RNA
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains.
  • LNPs a specific type of lipid- based carrier, are characterized as microscopic lipid particles typically having a solid core or partially solid core. Typically, an LNP does not comprise an interior aqua space sequestered from an outer medium by a bilayer.
  • the nucleic acid e.g. RNA
  • the nucleic acid may be encapsulated or incorporated in the lipid portion.
  • the lipid-based carriers are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes, nanoliposomes.
  • LNPs lipid nanoparticles
  • the lipid-based carriers are LNPs.
  • the lipid-based carriers encapsulate the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets).
  • the lipid-based carriers - encapsulated nucleic acid e.g. RNA
  • nucleic acid e.g. RNA
  • incorporation as the nucleic acid (e.g. RNA) is preferably contained within the interior of the lipid-based carriers.
  • the purpose of incorporating or encapsulating nucleic acid into lipid-based carriers may be to protect the nucleic acid from an environment which may contain enzymes, chemicals, or conditions that degrade the nucleic acid (e.g. RNA).
  • nucleic acid e.g. RNA
  • lipid-based carriers may promote the uptake of the nucleic acid and their release from the endosomal compartment, and hence, may enhance the therapeutic effect of the nucleic acid (e.g. RNA) when administered to a cell or a subject.
  • nucleic acid e.g. RNA
  • nucleic acids and/or nucleic acid sets (e.g. RNA, RNA sets) encoding antibodies are coformulated in lipid-based carriers.
  • nucleic acids and/or nucleic acid sets (e.g. RNA, RNA sets) encoding different antibodies are formulated in separate lipid-based carriers. In that context, LNPs are preferred.
  • the lipid-based carriers comprise at least one or more lipids selected from at least one aggregationreducing lipid, at least one cationic lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog, or any combinations thereof.
  • the lipid-based carriers comprise (i) an aggregationreducing lipid, (ii) a cationic lipid or ionizable lipid, and (iii) a neutral lipid/phospholipid or a steroid/steroid analog.
  • the lipid-based carriers comprise an (i) aggregation-reducing lipid, (ii) a cationic lipid or ionizable lipid, (iii) a neutral lipid or phospholipid, (iv) and a steroid/steroid analog.
  • Cationic lipids :
  • the lipid-based carriers comprise at least one cationic or cationizable lipid.
  • the cationic or ionizable lipid of the lipid-based carriers may be cationisable or ionizable, i.e. it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the lipid-based carriers comprise a cationic or ionizable lipid that preferably carries a net positive charge at physiological pH, more preferably the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group. Accordingly, in preferred embodiments, the lipid-based carriers comprise a cationic or ionizable lipid selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
  • cationic or ionizable lipids may be selected from the lipids disclosed in WO2018078053 (i.e. lipids derived from formula I, II, and III of WO2018078053, or lipids as specified in claims 1 to 12 of WO2018078053), the disclosure of WO2018078053 hereby incorporated by reference in its entirety.
  • lipids disclosed in Table 7 of WO2018078053 e.g. lipids derived from formula 1-1 to 1-41
  • lipids disclosed in Table 8 of WO2018078053 e.g. lipids derived from formula 11-1 to II-36
  • the lipid-based carriers comprise a cationic lipid selected or derived from structures 111-1 to HI-36 of Table 9 of published PCT patent application WO2018078053. Accordingly, formula 111-1 to HI-36 of WO2018078053, and the specific disclosure relating thereto, are herewith incorporated by reference. In preferred embodiments, the lipid-based carriers comprise a cationic lipid selected or derived from formula I H-3 of
  • a preferred lipid of formula III-3 has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1- diyl)bis(2-hexyldecanoate), also referred to as ALC-0315 i.e. CAS Number 2036272-55-4.
  • Further suitable cationic lipids may be selected or derived from cationic lipids according to claims 1 to 14 or table 1 of WO2021123332, the disclosure relating to claims 1 to 14 or table 1 of WO2021123332 herewith incorporated by reference.
  • suitable cationic lipids may be selected or derived from cationic lipids according compound 1 to compound 27 (C1 - C27) of table 1 of WO2021123332.
  • the lipid-based carriers comprise a cationic lipid selected or derived from (COATSOME®SS-EC) SS-33/4PE-15 (see C23 in table 1 of WO2021123332).
  • the lipid-based carriers comprise a cationic lipid selected or derived from HEXA-C5DE-PipSS (see C2 in Table 1 of WO2021123332).
  • the lipid-based carriers comprise a cationic lipid selected or derived from compound C26 as disclosed in table 1 of WO2021123332.
  • the lipid-based carriers comprise a cationic lipid selected or derived from 9-Heptadecanyl 8- ⁇ (2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate, also referred to as SM-102.
  • Other preferred lipid-based carriers e.g. LNPs
  • LNPs comprise a squaramide ionizable amino lipid, more preferably a cationic lipid selected from the group consisting of formulas M1 and M2 of US10392341B2, wherein the substituents (e.g. R1, R2, R3, R5, R6, R7, R10, M, M1, m, n, o, I) are defined in claims 1 to 13 of US10392341 B2; US10392341 B2 being incorporated herein in its entirety.
  • the at least one cationic or cationizable lipid is selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS, C26 (see C26 in Table 1 of WO2021123332).
  • the at least one cationic or cationizable lipid is selected from SS-33/4PE-15, HEXA-C5DE-PipSS, C26.
  • the cationic lipid as defined herein is present in the lipid-based carriers in an amount from about 20mol% to about 95mol%, relative to the total lipid content of the lipid-based carriers. In some embodiments, the cationic lipid is present in the lipid-based carriers in an amount from about 20 to about 75 mole percent. In embodiments, the cationic lipid is present in the lipid-based carriers in an amount from about 40 to about 60 mole percent. In other embodiments, the cationic lipid is present in the lipid-based carriers in an amount from about 55 to about 65 mole percent (based upon 100% total moles of lipid in the lipid nanoparticle). If more than one cationic lipid is incorporated within the lipid-based carriers, such percentages apply to the combined cationic lipids.
  • the ratio of cationic lipid to nucleic acid, preferably to RNA is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.
  • the lipid-based carriers comprise at least one aggregation reducing lipid or moiety.
  • aggregation reducing moiety refers to a molecule comprising a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
  • aggregation reducing lipid refers to a molecule comprising both a lipid portion and a moiety for reducing or preventing aggregation of the lipid-based carriers.
  • the lipid-based carriers may undergo charge-induced aggregation, a condition which can be undesirable for the stability of the lipid-based carriers. Therefore, it can be desirable to include a compound or moiety which can reduce aggregation, for example by sterically stabilizing the lipid-based carriers.
  • Such a steric stabilization may occur when a compound having a sterically bulky but uncharged moiety that shields or screens the charged portions of a lipid-based carriers from close approach to other lipid-based carriers.
  • stabilization of the lipid-based carriers is achieved by including lipids which may comprise a lipid bearing a sterically bulky group which, after formation of the lipid-based carrier, is preferably located on the exterior of the lipid-based carrier.
  • Suitable aggregation reducing groups include hydrophilic groups, e.g. monosialoganglioside GM1, polyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g., polyethylene glycol) or polypropylene glycol).
  • the lipid-based carriers comprise at least one aggregation reducing lipid.
  • the aggregation reducing lipid is selected from a polymer-conjugated lipid.
  • Lipids comprising a polymer as aggregation reducing group are herein referred to as “polymer conjugated lipid”.
  • the term “polymer conjugated lipid” refers to a molecule comprising both a lipid and a polymer portion, wherein the polymer is suitable of reducing or preventing aggregation of lipid-based carriers.
  • a polymer has to be understood as a substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits.
  • a suitable polymer may be a hydrophilic polymer.
  • An example of a polymer conjugated lipid is a PEGylated or PEG-conjugated lipid.
  • the polymer conjugated lipid is a PEG-conjugated lipid (or PEGylated lipid, PEG lipid).
  • the average molecular weight of the PEG moiety in the PEG- conjugated lipid preferably ranges from about 500 to about 8,000 Daltons (e.g., from about 1 ,000 to about 4,000 Daltons).
  • the PEG-conjugated lipid is selected or derived from 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG- PEG 2000).
  • DMG-PEG 2000 is typically considered a mixture of 1 ,2-DMG PEG2000 and 1 ,3- DMG PEG2000 in ⁇ 97:3 ratio.
  • the polymer conjugated lipid e.g. the PEG-conjugated lipid
  • the PEG-conjugated lipid is selected or derived from C10-PEG2K, or Cer8-PEG2K.
  • the PEG-conjugated lipid is selected or derived from formula (IV) of WO2018078053, preferably selected or derived from formula (IVa) of WO2018078053.
  • a PEG-conjugated lipid selected or derived from formula IVa may have the chemical term 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.
  • the at least one aggregation-reducing lipid, preferably the PEG-conjugated lipid is selected or derived from ALC-0159, DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K.
  • the lipid-based carriers comprise an aggregation reducing lipid, wherein the aggregation reducing lipid is not a PEG-conjugated lipid.
  • the aggregation reducing lipid may suitably be selected from a PEG-free lipid, e.g. a PEG-free polymer conjugated lipid.
  • the lipid-based carriers comprise an aggregation reducing lipid, wherein the aggregation reducing lipid is not a PEG-conjugated lipid.
  • the aggregation reducing lipid (or polymer conjugated lipid) is a PEG-free lipid that comprises a polymer different from PEG.
  • the lipid-based carriers do not comprise PEG.
  • a PEG-free lipid may be selected or derived from a POZ-lipid.
  • the POZ lipids or respectively preferred polymer conjugated lipids are described in WO2023031394, the disclosure herewith incorporated by reference.
  • the disclosure relating to polymer conjugated lipids of claims 1 to 8 and the disclosure relating to polymer conjugated lipids of claims 9 to 46 of W02023031394 are incorporated by reference.
  • the polymer conjugated lipid is a POZ-lipid, which is defined as a compound according to formula (POZ): [H] - [linker] - [M] , wherein
  • [H] is a homopolymer moiety comprising at least one polyoxazoline (POZ) monomer unit wherein R is C1 -9 alkyl or C2-9 alkenyl and n has a mean value ranging from 2 to 200, preferably from 20 to 100, more preferably from 24 to 26 or 45 to 50; [linker] is an optional linker group, and [M] is a lipid moiety.
  • POZ polyoxazoline
  • [H] is a heteropolymer moiety or homopolymer moiety comprising multiple monomer units selected from the group consisting of poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly(2-propyl-2- oxazoline) (PPOZ), poly(2-butyl-2-oxazoline) (PBOZ), poly(2-isopropyl-2-oxazoline) (PIPOZ), poly(2-methoxymethyl-2- oxazoline) (PMeOMeOx), and poly(2-dimethylamino-2-oxazoline) (PDMAOx), preferably wherein [H] is a homopolymer moiety comprising multiple PMOZ or PEOZ monomer units, more preferably wherein [H] comprises or preferably consists of multiple PMOZ monomer units, wherein (i) n has a mean value ranging from 2 to 200, preferably from 20 to 100, more
  • [H] is a heteropolymer moiety or homopolymer moiety comprising multiple monomer units selected from the group consisting of PmeOx, PETOx, PnPrOx, PcPrOx, PiPrOx, PsecBuOx, PiBuOx, PnBuOx, PPentOx, PheptOx, PNOx, PPheOx, PButEnOx, PPynOx, PDecEnOx, PiPrEnOx, and PIPOx (respective chemical structures are provided in W02023031394).
  • [M] as shown in formula (POZ) comprises at least one straight or branched, saturated or unsaturated alkyl chain containing from 6 to 30 carbon atoms, preferably wherein [M] comprises at least one straight or branched saturated alkyl chain, wherein the alkyl chain is optionally interrupted by one or more biodegradable group(s) and/or optionally comprises one terminal biodegradable group, wherein the biodegradable group is selected from the group consisting of but not limited to a pH-sensitive moiety, a zwitterionic linker, non-ester containing linker moieties and ester-containing linker moieties, amido, disulfide, carbonyl, ether, thioether, oxime, carbamate, urea, succinyl, succinamidyl, carbonate, succinoyl, phosphate esters, cyclic compound, heterocyclic compound, piperidine, pyrazine, pyridine, piperazine, and s
  • [linker] as shown in formula (POZ) is selected from the group consisting of but not limited to a pH-sensitive moiety, a zwitterionic linker, non-ester containing linker moieties and ester-containing linker moieties ( — C(O)O — or — OC(O) — ), amido, disulfide, carbonyl, ether, thioether, oxime, carbamate, urea, succinyl, succinamidyl, carbonate, succinoyl, phosphate esters, and sulfonate esters, as well as combinations thereof, wherein R3, R4 and R5 are, independently H or alkyl (e g. C1-C4 alkyl).
  • the polymer conjugated lipid is selected or derived from PMOZ 1 , PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 of W02023031394. In a very preferred embodiment in the context of POZ-lipids, the polymer conjugated lipid is selected or derived from PMOZ 4 of W02023031394.
  • the linker group [linker] comprises preferably an amide linker moiety. In a further very preferred embodiment in the context of POZ-lipids, the linker group [linker] comprises preferably an ester linker moiety. In a further very preferred embodiment in the context of POZ-lipids, the linker group [linker] comprises preferably a succinate linker moiety. In another very preferred embodiment in the context of POZ-lipids, the linker group [linker] comprises both an ester linker and an amid linker moiety. In another preferred embodiment, the linker group [linker] comprises both an ester linker, an amine linker and an amid linker moiety.
  • the polymer conjugated lipid does not comprise sulphur (S) or a sulphur group.
  • the at least one aggregation-reducing lipid is a PEG-conjugated lipid selected or derived from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K, or a POZ-lipid as defined herein.
  • lipid-based carriers include less than about 3, 2, or 1 mole percent of aggregation reducing lipid, based on the total moles of lipid in the lipid-based carrier.
  • lipid-based carriers comprise from about 0.1 % to about 10% of the aggregation reducing lipid on a molar basis, e.g. , about 0.5 to about 10%, about 0.5 to about 5% on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).
  • lipid- based carriers comprise from about 1.0% to about 2.0% of the aggregation reducing lipid on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).
  • lipid-based carriers comprise about 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of the aggregation reducing lipid on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).
  • the molar ratio of the cationic lipid to the aggregation reducing lipid ranges from about 100: 1 to about 25: 1.
  • Steroids, steroid analogues or sterols Steroids, steroid analogues or sterols:
  • the lipid-based carriers comprise a steroid, steroid analogue or sterol.
  • the steroid, steroid analogue or sterol may be derived or selected from cholesterol, cholesteryl hemisuccinate (CHEMS), and a derivate thereof.
  • the lipid-based carriers comprise a steroid, steroid analogue or sterol derived from a phytosterol (e.g., a sitosterol, such as beta-sitosterol), preferably from a compound having the structure of Formula I as disclosed in claim 1 of WG2020061332; the disclosure of W02020061332, especially the disclosure of Formula I and phytosterols being incorporated herewith by reference.
  • a phytosterol e.g., a sitosterol, such as beta-sitosterol
  • the steroid is an imidazole cholesterol ester or ”ICE” as disclosed in paragraphs [0320] and [0339]-[0340] of WO2019226925A1 ; WO2019226925A1 being incorporated herein by reference in its entirety.
  • the lipid-based carriers comprise cholesterol.
  • the molar ratio of the cationic lipid to cholesterol in the lipid-based carriers may be in the range from about 2:1 to about 1:1.
  • the lipid-based carrier comprises 10mol% to 60mol% or 25mol% to 40mol% sterol (based on 100% total moles of lipids).
  • the lipid-based carriers comprise at least one neutral lipid or phospholipid.
  • neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Suitable neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the bloodstream.
  • the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20.
  • neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used.
  • neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • the lipid-based carriers comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPO, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, transDOPE, 1 ,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (DPhyPE), or mixtures thereof.
  • the lipid-based carriers comprise a neutral lipid selected or derived from DSPC, DHPC, or DPhyPE.
  • the neutral lipid of the lipid-based carriers is selected or derived from DPhyPE.
  • the molar ratio of cationic lipid to neutral lipid in the lipid-based carriers ranges from 2:1 to 8:1.
  • the neutral lipid is preferably from about 5mol% to about 90mol% or about 5mol% to about 10mol% of the total lipid present in the lipid-based carrier.
  • the lipid-based carrier includes from about 0% to about 15% or 45% on a molar basis of neutral lipid, e.g., from about 3% to about 12% or from about 5% to about 10%.
  • Lipid-based carrier compositions are Lipid-based carrier compositions:
  • the lipid-based carriers comprise nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets) as defined herein, a cationic lipid as defined herein, an aggregation reducing lipid as defined herein, optionally, a neutral lipid as defined herein, and, optionally, a steroid or steroid analogue as defined herein.
  • nucleic acids and/or nucleic acid sets e.g. RNA or RNA sets
  • a cationic lipid as defined herein
  • an aggregation reducing lipid as defined herein
  • optionally, a neutral lipid as defined herein optionally, a steroid or steroid analogue as defined herein.
  • the lipid-based carriers comprise
  • the lipid-based carriers comprise
  • At least one cationic lipid selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS or C26;
  • lipid-based carriers encapsulate comprising the nucleic acids and/or nucleic acid sets (e.g. RNA or RNA sets).
  • the cationic lipids (as defined herein), neutral lipid (as defined herein), steroid or steroid analogue (as defined herein), and/or aggregation reducing lipid (as defined herein) may be combined at various relative ratios.
  • the lipid-based carriers comprise (i) to (iv) in a molar ratio of about 20-60% cationic lipid or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid, preferably wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
  • the ratio of cationic lipid or ionizable lipid to neutral lipid to steroid or steroid analogue to aggregation reducing lipid may be between about 30-60:20-35:20-30:1-15, or at a ratio of about 40:30:25:5, 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3 or 40:33:25:2, respectively.
  • the lipid-based carriers preferably the LNPs, preferably comprises 59mol% of cationic lipid “C26” (described herein above and/or below), 10mol% DPhyPE, 28.5mol% cholesterol and 2.5mol% of a POZ lipid, e.g. PMOZ 4 (described herein above and/or below).
  • the lipid-based carriers preferably the LNPs comprising the at least one nucleic acid, preferably the at least one RNA, comprise (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analogue selected from cholesterol; and (iv) at least one aggregation reducing lipid selected from ALC-0159, preferably wherein i) to (iv) are in a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analogue, and about 1.7% aggregation reducing lipid, preferably wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
  • the nucleic acid e.g. the RNA
  • the wt/wt ratio of lipid to nucleic acid (e.g. RNA) in the lipid-based carrier is from 10:1 to 60:1 , e.g. 40:1. In embodiments, the wt/wt ratio of lipid to nucleic acid is from 20:1 to 30:1 , e.g. 25:1. In other embodiments, the wt/wt ratio of lipid to nucleic acid is in the range of 20 to 60, preferably from 3 to 15, 4 to 8 or from 7 to 11.
  • the amount of lipid comprised in the lipid-based carriers may be selected taking the amount of the nucleic acid cargo into account. In one embodiment, these amounts are selected such as to result in an N/P ratio of the lipid-based carriers encapsulating the nucleic acid in the range of about 0.1 to about 50.
  • the N/P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid to the phosphate groups (“P”) of the nucleic acid which is used as cargo.
  • the N/P ratio may be calculated on the basis that, e.g., 1 pg nucleic acid typically contains about 3nmol phosphate residues, provided that the nucleic acid exhibits a statistical distribution of bases.
  • the “N”-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and, if present, cationisable groups.
  • the N/P ratio is in a range of 1 to 50. In preferred embodiments, the range is 1 to 20, e.g. about 6 or about 14 or about 17.
  • the composition comprises lipid-based carriers (encapsulating nucleic acid, preferably RNA) that have a defined size (particle size, homogeneous size distribution).
  • the size of the lipid-based carriers is typically described herein as Z-average size.
  • the terms “average diameter”, “mean diameter”, “diameter” or “size” for particles (e.g. lipid-based carriers) are used synonymously with the value of the Z-average.
  • Z-average size refers to the mean diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless.
  • DLS dynamic light scattering
  • the lipid-based carriers encapsulating the nucleic acid have a Z-average size ranging from about 50nm to about 200nm, preferably in a range from about 50nm to about 150nm, more preferably from about 50nm to about 120nm.
  • the polydispersity index (PDI) of the lipid-based carriers is typically in the range of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2.
  • 80% of the nucleic acid (e.g. RNA) comprised in the composition is encapsulated in lipid-based carriers, preferably 85% of the nucleic acid (e.g. RNA) comprised in the composition is encapsulated in lipid-based carriers, more preferably 90% of the nucleic acid (e.g. RNA) comprised in the composition is encapsulated in lipid- based carriers, most preferably 95% of the nucleic acid (e.g. RNA) comprised in the composition is encapsulated in lipid-based carriers.
  • the percentage of encapsulation may be determined by a RiboGreen assay as known in the art.
  • the lipid-based carriers have been prepared according to procedures described in WO2015199952, W02017004143 and WO2017075531, the full disclosures of which are incorporated herein by reference.
  • the lipid-based carriers as defined herein, preferably the LNPs encapsulate the nucleic acid, preferably the RNA, and have a Z-average size ranging from about 50nm to about 200nm.
  • the composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor (e.g. Toll-like receptor).
  • a RNA sensing pattern recognition receptor e.g. Toll-like receptor
  • Such an antagonist may preferably be co-formulated in lipid-based carriers as defined herein.
  • Suitable antagonist of at least one RNA sensing pattern recognition receptor are disclosed in WO2021028439, the full disclosure herewith incorporated by reference.
  • the disclosure relating to suitable antagonist of at least one RNA sensing pattern recognition receptors as defined in any one of the claims 1 to 94 of WO2021028439 are incorporated.
  • the composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor is a Toll-like receptor antagonist, preferably TLR7 and/or TLR8 antagonist.
  • the at least one antagonist of at least one RNA sensing pattern recognition receptor is selected from a nucleotide, a nucleotide analogue, a nucleic acid, a peptide, a protein, a small molecule, a lipid.
  • the at least one antagonist of at least one RNA sensing pattern recognition receptor is a single stranded oligonucleotide, preferably a single stranded RNA oligonucleotide.
  • the antagonist of at least one RNA sensing pattern recognition receptor is a single stranded oligonucleotide that comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85-212 of WO2021028439, or fragments of any of these sequences. Particularly preferred in that context is 5’-GAG CGmG CCA-3’ (SEQ ID NO: 85 of WO2021028439), or a fragment thereof.
  • the composition is a lyophilized (preferably carried out according to WO2016165831), a spray-dried (preferably carried out according to WO2016184575), or a spray-freeze dried composition (preferably carried out according to WO2016184576), optionally comprising at least one lyoprotectant (e.g. sucrose and/or trehalose)
  • a lyoprotectant e.g. sucrose and/or trehalose
  • the present invention relates to a combination for expression of one antibody, preferably for expression of one or more than one or a plurality of different antibodies in a cell or a subject.
  • nucleic acid of the first aspect may likewise be applied to the combination of the present aspect.
  • features and embodiments described in the context of the combination of the fifth aspect may likewise be applied to the nucleic acid of the first aspect, the nucleic acid set of the second aspect, the antibody of the third aspect, the composition of the fourth aspect, and any further aspect.
  • the combination comprises
  • the combination comprises
  • the combination comprises at least one or more than one compositions as defined in the context of the fourth aspect, preferably 2, 3, 4, 5, 6, 7, 8, or even more compositions, wherein each of the 2, 3, 4, 5, 6, 7, 8, or even more compositions provides the nucleic acid sequences for 2, 3, 4, 5, 6, 7, 8, or even more different antibodies each comprising at least one antibody heavy chain or HC element that comprises a different homodimerization promoter.
  • the components of the combination are formulated separately, preferably wherein the separate formulations are characterized by any of the features as defined in the context of the fourth aspect (see “Formulation and complexation features and embodiments”).
  • the components of the combination are formulated separately in LNPs as defined herein.
  • the separate formulations of the combination are administered in a spatially separated and/or timely staggered manner, e.g. to further reduce antibody misassembly.
  • administration of the combination to a cell or to a subject leads to production of at least two assembled antibodies in said cell or subject, preferably wherein at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the produced at least two antibodies are (correctly) assembled antibodies.
  • the present invention relates to a kit or kit of parts for expression of one antibody, preferably for expression of one or more than one or a plurality of different antibodies in a cell or a subject.
  • nucleic acid of the first aspect may likewise be applied to the kit or kit of parts of the present aspect.
  • nucleic acid set of the second aspect may likewise be applied to the kit or kit of parts of the present aspect.
  • the antibody of the third aspect may likewise be applied to any further aspect of the invention.
  • the kit or kit of parts comprises
  • the kit or kit of parts additionally comprises at least one liquid vehicle for solubilising.
  • the kit or kit of parts additionally comprises technical instructions providing information on administration and dosage of the kit components.
  • the kit or kit of parts may be applied e.g. for any of the applications or medical uses mentioned herein.
  • the kit or kit of parts comprise a syringe, an injection needle, a microneedle, an injection device, a catheter, an implant delivery device, or a micro cannula, or an inhalation device. Any of the above kits may be used in applications or medical uses or medical treatments as defined in the context of the invention.
  • nucleic acid nucleic acid set, antibody, composition, combination, and kit or kit of parts.
  • nucleic acid of the first aspect may likewise be applied to the medical uses of the current aspects.
  • features and embodiments described in the context of the medical uses of the present aspect may likewise be applied to any further aspect.
  • embodiments and features described herein in the context of the “medical use” or “further medical use” are also applicable to method of treatments as further outlined below.
  • embodiments and features described in the context of the “method of treatment’ are also applicable to first medical use and the further medical uses as described herein.
  • the invention provides a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect, for use as a medicament.
  • nucleic acid, nucleic acid set, antibody, composition, combination, or kit or kit of parts may be used for human medical purposes and/or for veterinary medical purposes, preferably for human medical purposes.
  • nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts may be advantageously used for human medical purposes and/or for veterinary medical purposes, preferably for human medical purposes, as the provided nucleic acid sequences generate at least two, preferably multiple correctly assembled antibodies.
  • the fact that upon administration, correctly assembled antibodies are produced may advantageously ensure efficacy and reduce the risk of unwanted side effects (due to off-target binding of misassembled antibody species).
  • nucleic acid, nucleic acid set, antibody, composition, combination, or kit or kit of parts may be used for the treatment, prophylaxis or therapy of any disorder, disease, or condition which can be treated or prevented by use of an antibody, in particular cancer, cardiovascular diseases, neurological diseases, infectious diseases, autoimmune diseases, virus diseases, bacterial diseases, genetic diseases or disorder and diseases or disorders related thereto.
  • the invention provides a nucleic acid of the first aspect, a nucleic acid set of the second aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect, for use in treatment or prophylaxis of a disease, disorder, or condition, wherein administration to a subject leads to expression of at least one therapeutically active antibody, preferably at least two or more therapeutically active antibodies in the subject.
  • the invention provides a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect, for use in the treatment or prophylaxis of an infection with a pathogen, for use in the treatment or prophylaxis of a cardiovascular disease, for use in the treatment or prophylaxis of a neurological disease, for use in the treatment or prophylaxis of an infectious disease, for use in the treatment or prophylaxis of an autoimmune diseases, for use in the treatment or prophylaxis of a cancer or tumour disease, for use in the treatment or prophylaxis of an eye or ophthalmic disease, for use in the treatment or prophylaxis of a lung or pulmonary disease, for use in the treatment or prophylaxis of a neurological disease, or for use in the treatment or prophylaxis of a genetic disease.
  • cancer 4 refers to the broad class of disorders and malignancies characterized by hyper proliferative cell growth, either in vitro (e.g., transformed cells) or in vivo.
  • Conditions which can be treated or prevented by the compositions and methods of the invention include, e.g., a variety of neoplasms, including benign or malignant tumours, a variety of hyperplasias, or the like.
  • Compositions and methods of the invention can achieve the inhibition and/or reversion of undesired hyper proliferative cell growth involved in such conditions.
  • Infectious diseases are typically caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi. Infectious diseases can usually be spread, directly or indirectly, from one person to another.
  • cardiovascular disease typically includes any disorders/diseases of the cardiovascular system.
  • cardiovascular diseases include coronary heart disease, arteriosclerosis, apoplexy and hypertension.
  • neurological disease typically includes disorders/diseases of the nervous system.
  • neurological diseases include Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease.
  • autoimmune disease typically refers to a pathological state rising from an abnormal immune response of the body to substances and tissues that are normally present in the body.
  • the invention provides a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use in the treatment or prophylaxis of an infection with a pathogen (e.g. passive vaccination), preferably wherein the pathogen is a virus or a bacterium.
  • a pathogen e.g. passive vaccination
  • the invention provides a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use in the treatment or prophylaxis of a cancer or tumour disease.
  • administration of the nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts to a cell or to a subject leads to expression of at least two assembled antibodies in said cell or subject, wherein, preferably, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the expressed at least two antibodies are (correctly) assembled antibodies.
  • mass spectrometry MS can be used to determine the percentage of assembled antibodies and misassembled antibodies.
  • the invention relates to a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a chronic medical treatment.
  • chronic medical treatment relates to treatments that require the administration more than once, for example once or more than once a day, once or more than once a week, once or more than once a month.
  • applying or administering of the nucleic acid, nucleic acid set, antibody, composition, combination, or kit or kit is performed more than once, for example once or more than once a day, once or more than once a week, once or more than once a month (as defined herein).
  • parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intraocular, intravitreal, subretinal, intratumoral.
  • the step of applying or administering is subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intraocular, intravitreal, subretinal, intranasal or intratumoral.
  • the step of applying or administering is intravenous, intramuscular or intrapulmonal.
  • the step of applying or administering is intravenous.
  • the step of applying or administering may be at different injection sites for each entity.
  • the step of applying or administering may be at a different injection regimen or time-staggered. That procedure may improve the correct assembly of antibodies in vivo as each antibody (provided by a nucleic acid or nucleic acid set) may be administered as a separate entity.
  • applying or administering of the nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts leads to expression of at least two assembled antibodies, wherein said at least two assembled antibodies are detectable at least about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 96 hours, 120 hours, 144 hours, 156 hours, 168 hours, or 180 hours post-administration (e.g., post single administration).
  • applying or administering of the nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts leads to expression of at least two assembled antibodies, wherein said at least two assembled antibodies are detectable at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 22 days, 25 days, or 30 days post-administration (e.g., post single administration).
  • applying or administering of the nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts leads to expression of at least two assembled antibodies, wherein said at least two assembled antibodies are detectable at least about 0.5 weeks, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, or 8 weeks postadministration (e.g., post single administration).
  • the systemic expression of the antibody is detectable at least about 1 month, 2 months, 3 months, or 4 months post-administration (e.g., post single administration).
  • applying or administering of the nucleic acid, nucleic acid set, composition, combination, or kit or kit of parts leads to expression of at least two assembled antibodies in target cells or tissues, wherein said target cells or tissues may be selected from hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes,
  • a further aspect of the present invention relates to a method of treating or preventing a disease, disorder, or condition.
  • nucleic acid of the first aspect the nucleic acid set of the second aspect, the antibody of the third aspect, the composition of the fourth aspect, the combination of the fifth aspect, or the kit or kit of parts of the sixth aspect may likewise be applied to the methods of treatment of the current aspects.
  • features and embodiments described in the context of the method of treatment of the present aspect may likewise be applied to any further aspect.
  • embodiments described above in the context of the first medical use and the further medical uses) are also applicable to methods of treatment as described herein and vice versa.
  • compositions, nucleic acid set, combination and/or kit or kit of parts may be used in a method for human medical purposes and/or for veterinary medical purposes, preferably for human medical purposes.
  • the invention provides a method of treating or preventing a disorder or condition, wherein the method comprises applying or administering to a subject in need thereof a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect.
  • the nucleic acid, nucleic acid set, antibodies, composition, combination, or kit or kit of parts may be used in a method of treating or preventing a disorder or condition, wherein the disorder or condition can be any disorder, disease, or condition which can be treated or prevented by use of an antibody, in particular cancer, cardiovascular diseases, neurological diseases, infectious diseases, autoimmune diseases, virus diseases, bacterial diseases, genetic diseases or disorder and diseases or disorders related thereto.
  • the disorder or condition can be any disorder, disease, or condition which can be treated or prevented by use of an antibody, in particular cancer, cardiovascular diseases, neurological diseases, infectious diseases, autoimmune diseases, virus diseases, bacterial diseases, genetic diseases or disorder and diseases or disorders related thereto.
  • the invention provides a method of treating or preventing a disorder or condition, wherein the method comprises applying or administering to a subject in need thereof a nucleic acid of the first aspect, a nucleic acid set of the second aspect, an antibody of the third aspect, a composition of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect, wherein the disorder or condition is an infection with a pathogen, a cardiovascular disease or condition, a neurological disease or condition, an infectious disease or condition, an autoimmune diseases or condition, a cancer or tumour disease or condition, an eye or ophthalmic disease or condition, a lung or pulmonary disease or condition, a neurological disease or condition, a genetic disease or condition, or a lung disease or condition.
  • the disorder or condition is an infection with a pathogen, a cardiovascular disease, a neurological disease, an infectious disease, an autoimmune disease, a cancer or tumour disease, an eye or ophthalmic disease, a lung or pulmonary disease, a neurological disease, or a genetic disease.
  • administration to a cell or to a subject leads to expression of at least two assembled antibodies in said cell or subject, wherein, preferably, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the expressed at least two antibodies are (correctly) assembled antibodies.
  • mass spectrometry MS can be used to determine the percentage of assembled antibodies and misassembled antibodies.
  • the subject in need thereof is a mammalian subject, preferably a human subject.
  • the subject in need thereof is a young infant human subject, a newborn human subject, immunocompromised human subject, a pregnant human subject, a breast-feeding human subject, or an elderly human subject.
  • the subject in need is a mammalian subject, preferably a human subject.
  • the method of treatment is a chronic medical treatment. Accordingly, applying or administering is performed more than once, for example once or more than once a day, once or more than once a week, once or more than once a month (as defined herein).
  • a further aspect relates to a method expressing or producing at least one or at least two nucleic acid encoded antibodies in a cell, an organ, or a tissue.
  • nucleic acid of the first aspect the nucleic acid set of the second aspect, the composition of the fourth aspect, the combination of the fifth aspect, or the kit or kit of parts of the sixth aspect may likewise be applied to the method for expressing or producing of the present aspect.
  • features and embodiments described in the context of the methods for expressing or producing of the present aspect may likewise be applied to any fu rther aspect.
  • the method is for expressing or producing at least two nucleic acid encoded antibodies in a cell, organ, or tissue of a subject, comprising administering or applying to a subject a nucleic acid as defined in the context of the first aspect, a nucleic acid set as defined in the context of the second aspect, a composition as defined in the context of the fourth aspect, a combination as defined in the context of the fifth aspect, and/or a kit or kit of parts as defined in the context of the sixth aspect.
  • the invention provides a method of expressing at least one, preferably at least two nucleic acid-encoded antibodies in a cell, organ or tissue in a subject, wherein the method comprises applying or administering at least one nucleic acid of the first aspect, at least one nucleic acid set of the second aspect, at least one composition of the fourth aspect, at least one combination of the fifth aspect, and/or at least one the kit or kit of parts of the sixth aspect to a cell or a subject.
  • administering or applying leads to expression of at least two assembled antibodies in a cell, organ or tissue in a subject, wherein, preferably, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the expressed at least two antibodies are (correctly) assembled antibodies.
  • mass spectrometry MS can be used to determine the percentage of assembled antibodies and misassembled antibodies
  • the method does not involve a harvesting step and/or a purification step of the expressed at least one, preferably at least two antibodies (e.g. harvesting from a cell, e.g. a bacterium or a cell culture).
  • the method of expressing is an in vivo method for expressing at least one, preferably at least two correctly assembled antibodies.
  • the nucleic acid encoded antibodies are not provided by plasmid DNA. In preferred embodiments, the nucleic acid encoded antibodies are provided by RNA, preferably mRNA.
  • a further aspect relates to a method for the production of at least two nucleic acid encoded antibodies in a cell.
  • the invention provides a method of producing at least one, preferably at least two nucleic acid-encoded antibodies, wherein the method comprises a step of (i) applying or administering at least one nucleic acid of the first aspect, at least one nucleic acid set of the second aspect, at least one composition of the fourth aspect, at least one combination of the fifth aspect, and/or at least one kit or kit of parts of the sixth aspect to allow production of at least one, preferably at least two assembled antibodies in a cell, and, optionally, a step of (ii) isolating and/or purifying the produced assembled antibodies, wherein the method is an in vitro, in situ, or ex vivo method.
  • the nucleic acid sequences used in the method of producing are RNA sequences, preferably mRNA sequences.
  • administering or applying leads to expression of at least two assembled antibodies in said cell, wherein, preferably, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the expressed at least two antibodies are (correctly) assembled antibodies.
  • mass spectrometry MS can be used to determine the percentage of assembled antibodies and misassembled antibodies
  • the cell is a cell line suitable for the production of therapeutic antibodies.
  • a mammalian host cell line including (without limiting) NS0 murine myeloma cells, PER. C6® human cells, and Chinese hamster ovary (CHO) cells.
  • the cell is a yeast cell, or a bacterial cell.
  • the obtained produced antibodies may be isolated from the cells and may be purified using typical antibody purification methods known in the art (e.g. affinity purification, chromatography, filtration, centrifugation, dialysis etc.)
  • a further aspect relates to an a method for reducing the production of HC-HC by-products as defined herein in nucleic acid encoded antibody mixtures as defined herein for in vitro or in vivo applications by introducing different HC-HC homodimerization promoters into the respective heavy chains, preferably wherein the different homodimerization promoters are selected from Homo01 - Homol 3 and/or or Homo01 Homol 3', preferably selected from
  • Homo01 /03/06/09 and/or Homo01703705706709’ are examples of Homo01 /03/06/09 and/or Homo01703705706709’.
  • nucleic acid of the first aspect the nucleic acid set of the second aspect
  • composition of the fourth aspect the combination of the fifth aspect, the kit or kit of parts of the sixth aspect, the method of expressing, or the method of producing
  • features and embodiments described in the context of the nucleic acid of the first aspect, the nucleic acid set of the second aspect, the composition of the fourth aspect, the combination of the fifth aspect, the kit or kit of parts of the sixth aspect, the method of expressing, or the method of producing may likewise be applied to the method for reducing the production of HC-HC by-products of the present aspect.
  • a further aspect relates to a nucleic acid cassette encoding a homodimerization promoter.
  • the invention provides a nucleic acid cassette encoding a homodimerization promoter, wherein the homodimerization promoter is characterized by any of the features as defined in the context of the first aspect.
  • the homodimerization promoter comprises or consists of at least one or two assembly elements selected from assembly element (AE) 1 to 22. In preferred embodiments of the nucleic acid cassette, the homodimerization promoter comprises or consists of at least one or two assembly elements (AEs) selected from assembly element 1 , 4, 5, 8, 10 as defined herein. In particularly preferred embodiments of the nucleic acid cassette, the homodimerization promoter is selected or derived from Homo01- Homo13 or Homo01’- Homo13’, preferably selected from Homo01 /03/06/09 and/or Homo01703705706709’.
  • the nucleic acid cassette encodes for a homodimerization promoter that comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 96-108, 110-122, 124-136, or a fragment or variant thereof.
  • the nucleic acid cassette encodes for a homodimerization promoter that comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 222-234, or a fragment or variant thereof.
  • a further aspect relates to a homodimerization promoter amino acid sequence.
  • the invention provides a homodimerization promoter amino acid sequence, wherein the homodimerization promoter is characterized by any of the features as defined in the context of the first aspect.
  • the homodimerization promoter comprises or consists of at least one or two assembly elements selected from assembly element (AE) 1 to 22. In preferred embodiments in that context, the homodimerization promoter comprises or consists of at least one or two assembly elements (AEs) selected from assembly element 1 , 4, 5, 8, 10 as defined herein. In particularly preferred embodiments in that context, the homodimerization promoter is selected or derived from Homo01 - Homol 3 or Homo01 ’- Homol 3’, preferably selected from Homo01 /03/06/09 and/or Homo01703705706709’.
  • the homodimerization promoter comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 96-108, 110-122, 124-136, or a fragment or variant thereof.
  • the homodimerization promoter comprises or consists of an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the following SEQ ID NOs: 222- 234, or a fragment or variant thereof.
  • the invention provides a method of identifying and selecting a suitable homodimerization promoter for expression of an antibody comprising homodimeric antibody heavy chains or HC elements in a cell or subject, wherein the selection is based on energy difference calculations showing self-/homo-assembly of HC components is preferable (lower energy barrier) to hetero-assembly with wildtype (unmodified) antibody HCs.
  • the invention provides a method of identifying and selecting at least one suitable homodimerization promoter for expression of at least two antibodies comprising homodimeric antibody heavy chains or HC elements in a cell or subject, wherein the selection is based on energy difference calculations shewing self-/homo-assembly of each HC component is preferable (lower energy barrier) to hetero-assembly with any of the other mixture HC components.
  • an atomic model of the dimeric Fc is generated based on the available crystal structures of the respective dimeric Fc.
  • Options to disturb the interface may be identified by screening the interface for unfavourable sidechain exchanges and finding ways to accommodate such changes (e.g. opposing chain).
  • Existing concepts for bispecific-assembly may be taken into account by the skilled person.
  • a further step comprises dissecting the options to steer assembly and/or optimizing assembly using protein design software as commonly used in the art, preferably using protein design software such as CCG’s MOE.
  • protein design software such as CCG’s MOE.
  • Intended changes via mutations may be introduced in an energy-minimized dimeric Fc- model. The best candidates are rated based on the differences in calculated binding energy.
  • the obtained concepts may be combined to yield best candidates according to the assessment.
  • favourable energy values are determined as defined in the context of the first aspect, e.g. wherein the assembly of homodimeric antibody HC elements comprising the homodimerization promoter is characterized by a more favourable (lower) binding energy than the assembly of corresponding non-modified (wildtype) HC elements, preferably wherein the assembly of homodimeric antibody HC elements comprising the homodimerization promoter is additionally characterized by a more favourable (lower) binding energy than the assembly of the respective HC element to a different HC element, in particular a non-modified (wildtype) HC element.
  • homodimeric antibody HC elements comprising the homodimerization promoter are characterized by a negative (directed) assembly energy.
  • respective energy values of the method are determined according to formula 1 , 2, and 3 as defined in that context.
  • the identified homodimerization promoters are included into HC or HC elements, optionally encoded on a nucleic acid (e.g. an mRNA) and used for expressing at least one antibody.
  • a nucleic acid e.g. an mRNA
  • Figure 1 exemplifies the concept of the invention.
  • Figure 1 a shews the CH3-CH3 homodimer of an exemplary IgG that comprises two identical heavy chains (HC) carrying a homodimerization promoter in top view; the rest of the antibody is not shown.
  • the arrows pointing in opposite directions illustrate the parallel C2 symmetry.
  • the CH3-CH3 interface is formed by two identical CH3 domains comprising a (i.e. the same) homodimerization promoter, which is exemplarily made up of one knob modification and one hole modification.
  • the knob and hole modification of one homodimerization promoter can interact with the corresponding counterparts of the homodimerization promoter on the opposite CH3 domain in a parallel C2 symmetric manner.
  • the homodimerization promoters on both CH3 domains are the same (e.g. have the same amino acid sequence) and thus are compatible, interact, and promote specific assembly. Accordingly, the shown CH3-CH3 homodimer may belong to an exemplary IgG antibody comprising a homodimerization promoter in the context of the invention.
  • Figure 1b shows the CH3-CH3 heterodimer of an exemplary undesired IgG antibody that comprises a wild-type, non-modified HC lacking a homodimerization promoter (left CH3; dark grey) and a HC carrying a homodimerization promoter (right CH3; light grey); the rest of the antibody is not shown.
  • the arrows pointing in opposite directions again illustrate the parallel C2 symmetry.
  • the homodimerization promoter and the wildtype CH3 domain are not compatible, and thus are not able to interact and promote specific assembly. In feet, the opposite is the case: the interface is disturbed and the assembly prevented or reduced (indicated by the cross).
  • the HC configuration underlying Figure 1 b would lead to an undesired misassembled by-product that could theoretically occur if two such HC are co-expressed in the same cell. In the context of the invention, the formation of misassembled by-products is prevented or reduced.
  • Figure 2 illustrates the HC-HC assembly options for the nucleic acid (e.g. mRNA) encoded antibody mixtures of Example 3, comprising one antibody with a homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter.
  • nucleic acid e.g. mRNA
  • Figure 2 illustrates the HC-HC assembly options for the nucleic acid (e.g. mRNA) encoded antibody mixtures of Example 3, comprising one antibody with a homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter.
  • the AA configuration shows an assembled CH3-CH3 homodimer of an antibody comprising two wild-type (non-modified) HC lacking a homodimerization promoter
  • the BB configuration shows an assembled CH3-CH3 homodimer of an antibody comprising two HC comprising the same homodimerization promoter.
  • AA and BB are desired configurations, which is highlighted by the frames.
  • the other configuration AB shows a CH3-CH3 heterodimer of an undesired misassembled by-product that could theoretically occur if such HC are co-expressed in the same cell.
  • the formation of misassembled by-products is prevented or reduced (indicated by the cross) to allow co-expression of at least two correctly assembled antibodies in the same cell.
  • Figure 3 shows antibody levels detected by ELISA in supernatants from two cell-lines (Part 1 HeLA cells, Part 2 CHO cells) that were transfected in vitro with mRNA compositions encoding for anti-Hemolysin A antibodies comprising different homodimerization promoters (Homo01 -Homol 3). Results are depicted as mean and standard error of the mean (triplicate per condition). The experiment shows that all antibodies comprising a homodimerization promoter were produced and secreted into the cell supernatant upon mRNA transfection.
  • Figure 4 shows the relative amount of different antibody configurations detected by mass spectrometry in purified supernatants from HeLa cells that were transfected in vitro with mRNA compositions encoding for one anti-Hemolysin A full-length antibody comprising a homodimerization promoter and one full-length anti-Hemolysin A wild-type (nonmodified) antibody lacking a homodimerization promoter.
  • the theoretically possible antibody HC configurations of this experimental setup are further described in Figure 2 and Table E5. The experiment shews that only the desired antibody HC configurations and no misassembled antibody HC configuration were detected, n.a., not available (see further explanations hereto in Example 3); n.d., not detected.
  • Example 1 In silico studies and preparation of DNA and RNA constructs
  • homodimerization promoters 13 homodimerization promoters (HomoOl -Homol 3) were selected for subsequent experimental testing.
  • DNA sequences encoding the different antibody chains were prepared and used for subsequent RNA in vitro transcription reactions.
  • Said DNA sequences were prepared by modifying encoding DNA sequences by introducing a G/C optimized coding sequence for stabilization and expression optimization. Sequences were introduced into a pUC derived DNA vector to comprise stabilizing 5’-UTR and 3’-UTR sequences, a histone stem-loop and a stretch of adenosines.
  • the obtained plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA constructs were extracted, purified and enzymatically linearized using a restriction enzyme and the linear DNA was used for subsequent RNA in vitro transcription.
  • DNA plasmids prepared according to section 1.2 were used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7G(5’)ppp(5’)(2’OMeA)pG; TriLink) under suitable buffer conditions.
  • the obtained RNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; W02008077592). All RNA constructs used in the Examples complied with internal standards regarding e.g. RNA integrity, RNA purity, or capping degree.
  • the generated RNA sequences/constructs including the encoded proteins are provided in Tables 4-6 further below.
  • Example 4 mRNA constructs encoding for full-length anti-Hemolysin A antibodies are used as depicted in Table E1. As further depicted in Table E5 (0#) and Table E4, a wild-type (unmodified) antibody lacking a homodimerization promoter and 13 antibodies with 13 different homodimerization promoters, respectively, are encoded by one mRNA encoding for the common LC and one mRNA encoding for the respective HC chain. Accordingly, a fully and correctly assembled anti-Hemolysin A antibody in this context consists of two identical antibody LC and two identical antibody HC.
  • Table E1 Overview of mRNA constructs encoding for anti-Hemolysin A full-length antibodies in Examples 2-4
  • Example 5 For Example 5, optionally Example 4, one or more mRNA constructs encoding for anti-rabies antibodies in the scFv-Fc format as depicted in Table E2 may be used. Accordingly, a fully and correctly assembled anti-Rabies antibody in this context consists of two identical “VL - 15 aa linker - VH -Fc" chains.
  • Table E2 Overview of mRNA constructs encoding for anti-Rabies scFv-Fc antibodies in Examples 4-5
  • mRNA constructs encoding for anti-rabies scFv-Fc antibodies with heterodimerization promoter pairs as depicted in Table E3 may be used.
  • a fully assembled antibody consists of two “VL - 15 aa linker - VH -Fc" chains (chain A and B of the respective heterodimerization promoter pair).
  • Table E3 Overview of mRNA constructs encoding for anti-Rabies scFv-Fc antibodies with heterodimerization promoter pairs in Example 4
  • RNA IDs R8534, R8535, R8536, R8537 of WO2022023559 SEQ ID NOs: 92, 93, 96, 97 of WO2022023559, respectively; Composition 1 in Table 4 of WO2022023559) and/or RNA IDs R8534, R8536, R8537, R8538, R8539 of WO2022023559 (SEQ ID NOs: 92, 96, 97, 98, 99 of WO2022023559, respectively; Composition 5 in Table 6 of WO2022023559) may be used as positive controls for HC misassembly.
  • Example 2 In vitro expression analysis of antibodies comprising a homodimerization promoter
  • the goal of the experiment was to confirm that antibodies comprising homodimerization promoters are produced and secreted upon in vitro mRNA transfection.
  • ELISAs with supernatants from cells transfected in vitro with an mRNA composition encoding for a single full-length anti-Hemolysin A antibody with homodimerization promoter were performed.
  • HeLa (60000 cellsAvell) and HEK293T (75000 cells/well) cells were seeded in 24 well plates. 24 hours after seeding, cells were transfected with 500ng mRNA/well in triplicates for the respective compositions described in Table E4 (for mRNA constructs see furthermore Table E1). HC and LC mRNAs were mixed at a w/w ratio of 3:1 and Lipofectamine 2000 was used for transfection. 24 hours after transfection, supernatants were collected and subjected to further analysis.
  • Results are shown in Figure 3 and are depicted as mean and standard error of the mean (triplicate per condition). The experiment confirmed that all antibodies comprising a homodimerization promoter (13 different tested) are produced and secreted upon in vitro mRNA transfection.
  • Example 3 In vitro expression analysis of mRNA encoded antibody mixtures comprising one antibody with a homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter
  • homodimerization promoters allows for the production of two correctly assembled antibodies in the same cell, e.g. as addressed in this Example: one antibody comprising a homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter.
  • HeLa cells were seeded in T175 flasks (one per composition). After 24h, cells were transfected with 135pg of the respective mRNA compositions (as depicted in Table E5) and Lipofectamine 2000 as transfection reagent.
  • HC/LC mRNA were used at a mass ratio of 3:1 for composition 0# and HCwr/HCnomoxx/LC mRNA at a mass ratio of 3:3:2 for compositions 1 #-13#.
  • the produced and secreted antibodies were purified from the HeLa cell culture medium using FLPC (HiTrap MabSelect PrismA purification columns; Cytiva; #17-5498-52).
  • the purified antibody compositions complied with in-process-controls (Western Blots, data not shown) and were subjected to further analysis as outlined in
  • Mass spectrometry was performed as previously described in WO2022023559.
  • 12.5pg antibody sample was treated with 0.5pl PNGaseF (R&D Systems, #9109-GH) and incubated over night at 37°C to allow deglycosylation.
  • the sample was treated with 0.32pl cysteine protease FabALACTICA (Genovis, #A0-AG1-020) to digest the antibodies above the hinge-region into a Fab’ fragments and Fc-dimer fragments.
  • the enzymatic treatment reduced full-length antibodies (molecular weight of about 150kDa plus glycan pattern) to Fc-dimer fragments of about 50kDa (without glycan pattern).
  • the sample was analyzed using HPLC-MS as outlined below to determine the relative amounts of correctly assembled and misassembled Fc-dimers.
  • digested probe was chromatographically purified, desalted, and analyzed using RP-HPLC (Acquity BEH300 C4, 1mm 50mm, 1.7pm) coupled to MS (QTOF mass spectrometer, MAXIS, Bruker Daltonics). The massspectra for each sample were recorded and the individual mass spectra over the elution time of the Fc-dimers summed up and subsequently deconvolved by means of the MaxEnt algorithm.
  • compositions #1 , #6, #9 comprising mRNA encoding for a wild-type (non-modified) antibody and antibodies with homodimerization promoter Homo01 , Homo06, Homo09, respectively, led to an equal amount of both produced antibodies in the mixture.
  • Example 4 In vitro expression analysis of mRNA encoded antibody mixtures comprising two antibodies with a (different) homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter
  • homodimerization promoters allow for the production of three correctly assembled antibodies in the same cell, e.g. as addressed in this Example: two antibodies comprising a (different) homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter. Further, these in silico analyses indicated that three such correctly assembled antibodies can be produced without generating undesired by-products (caused e.g. by mismatching of antibody chains). Accordingly, these in in silico analyses pointed to the fact that more than one antibody with homodimerization promoter can be used in such antibody mixtures and that different homodimerization promoters in different antibodies can be combined with each other to promote specific antibody assembly.
  • homodimerization promoters HomoOl , Homo03, Homo06, Homo09 More specifically for the six possible combinations of two antibodies with each having one (different) homodimerization promoter from this list and one wildtype (unmodified) antibody (i.e. WT/Homo01/Homo03; WT/Homo01/Homo06; WT/Homo01/Homo09; WT/Homo03/Homo06; WT/Homo03/Homo09; WT/Homo06/Homo09).
  • WT/Homo01/Homo03 WT/Homo01/Homo06
  • WT/Homo01/Homo09 WT/Homo03/Homo06
  • WT/Homo03/Homo09 WT/Homo06/Homo09
  • mRNA constructs encoding for two antibodies comprising a (different) homodimerization promoter and for one wild-type (unmodified) antibody lacking a homodimerization promoter are analysed by mass spectrometry.
  • mRNA constructs encoding for full- length anti-Hemolysin A antibodies (all have a common LC) shown in Table E1 may be used.
  • mRNA constructs encoding for anti-rabies antibodies in the scFv-Fc format as depicted in Table E2 may be used.
  • one or more mRNA compositions encoding for the above cited particularly preferred combinations may be analyzed.
  • mRNA compositions for transfection are provided in Table E6 and Table E7 and are used as further outlined below. Also provided in these tables are the three desired HC-HC configurations and the 3 undesired HC-HC configurations of potential misassembled species or by-products.
  • Table E6 Overview of mRNA compositions in Examples 4/5 - anti-Hemolysin A full-length antibodies
  • Table E7 Overview ofmRNA compositions in Examples 4/5 - anti-Rabies scFv-Fc antibodies
  • compositions 1 in Table 4 of WO2022023559 or Composition 5 in Table 6 of WO2022023559 may be used. These compositions showed misassembled HC species in the past (see Table 5 and 7 of WO2022023559).
  • compositions based on anti-Rabies scFv-Fc antibodies shown in Table E8 may be analyzed.
  • HC/LC mRNA are used at a mass ratio of 3: 1 for composition 0‘, HCwr/HChomoxx/HCHomoYY/LC mRNA at a mass ratio of 1 : 1 : 1 : 1 for compositions 1 *- 6* and all scFv-Fc mRNA constructs at equal wt amounts (i.e. 1:1:1 or 1:1:1:1) for 1 ⁇ -6 ⁇ as well as 1$-2$.
  • the produced and secreted antibodies are purified from the HeLa cell culture medium using FLPC (HiTrap MabSelect PrismA purification columns; Cytiva; #17-5498-52).
  • the purified antibody compositions are tested for compliance with in-process-controls (Western Blots) and are subjected to further analysis as outlined in 4.2.
  • 12.5pg antibody sample (obtained in step 4.1) is further processed and analyzed according to section 3.2.
  • Example 5 In vivo expression analysis ofmRNA encoded antibody mixtures comprising two antibodies with a homodimerization promoter and one wild-type (unmodified) antibody lacking a homodimerization promoter
  • Antibody mixtures purified from mice that are administered with mRNA compositions encoding for two antibodies comprising a (different) homodimerization promoter and for one wild-type (unmodified) antibody lacking a homodimerization promoter are analysed by mass spectrometry.
  • the mRNA constructs encoding for anti-Rabies antibodies in the scFv-Fc format as shown in Table E2 may be used.
  • compositions as shown in Table E7 may be tested in the experiment and used as further outlined below. Also provided in Table E7 are the three desired HC-HC configurations and the 3 undesired HC-HC configurations of potential misassembled species or by-products. Alternatively, one or more compositions of Table E6 encoding for antiHemolysin A full-length antibodies may be analyzed.
  • Lipid nanoparticle formulation of mRNA constructs mRNA constructs are formulated in lipid nanoparticles (final mRNA concentration 0.2mg/ml; all constructs at a mass ratio of 1 : 1 : 1 ).
  • LNPs are e.g. prepared using i) the cationic lipid C26, (ii) the neutral lipid DphyPE, (iii) cholesterol, and (iv) a PEG-free aggregation reducing lipid, wherein (i) to (iv) are in a molar ratio of about 59% cationic lipid, 10% neutral lipid, 28.5% cholesterol, and 2.5% aggregation reducing lipid.
  • Lipid solution in ethanol
  • RNA solution in aqueous buffer
  • Obtained LNPs are re-buffered in a carbohydrate buffer via dialysis, and up- concentrated to a target concentration using TFF.
  • 2mg/kg of the respective LNP-formulated mRNA composition is injected intravenously into the tail vain of SCID mice. 48h after administration the animals are sacrificed, the blood is collected and the serum is prepared.
  • the produced and secreted antibodies are purified from mouse serum using FPLC as described in section 3.1.
  • the purified antibody mixture is subjected to further analysis as outlined in section 5.4.
  • 12.5pg antibody sample (obtained in step 5.3) is further processed and analyzed according to section 3.2.
  • an antibody mixture of up to 3 correctly assembled antibodies can be produced upon in vitro and/or in vivo administration of a nucleic acid composition of the invention.
  • nucleic acid compositions encoding a plurality of different assembled antibodies (e.g. 4, 5, 6, 7, 8, 9, 10, or more assembled antibodies).
  • nucleic acids encoding for antibodies with heterodimerization promoter pairs or more preferably single-chain antibodies (e.g. BiTE), which intrinsically do not need guided assembly measures, may further expand the number of antibodies in such antibody mixtures.
  • the data demonstrates that the production of a plurality of fully and correctly assembled antibodies can be accomplished by delivering a nucleic acid composition encoding said plurality of antibodies, wherein at least one coding sequence of the nucleic acid sequences encodes at least one homodimerization promoter.

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Abstract

La présente invention concerne, entre autres, un acide nucléique pour l'expression d'un anticorps comprenant des éléments d'anticorps homodimères à chaîne lourde (HC) dans une cellule ou chez un sujet, l'acide nucléique comprenant au moins une séquence de codage codant pour au moins un élément HC d'anticorps qui comprend un promoteur d'homodimérisation. En outre, l'invention concerne un ensemble d'acides nucléiques, un anticorps codé par l'acide nucléique, une composition, une combinaison et un kit ou un kit de pièces. De plus, l'invention concerne des première et seconde utilisations médicales, des méthodes de traitement ou de prévention de maladies, de troubles ou d'états, des procédés d'expression et de production. L'invention concerne en outre une cassette d'acide nucléique codant pour un promoteur d'homodimérisation, un promoteur d'homodimérisation en tant que tel, et des procédés d'identification et de sélection de promoteurs d'homodimérisation appropriés pour l'expression d'un ou de plusieurs anticorps codés par un acide nucléique.
PCT/EP2023/067201 2023-06-23 2023-06-23 Anticorps codés par un acide nucléique Pending WO2024260570A1 (fr)

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