Engineered ch1 and CL domains for the prevention of chain mispairing

Abstract

The present invention is concerned with antibodies or fragments thereof as well as fusion proteins comprising the same, and nucleic acids encoding the afore-mentioned antibodies or fragments thereof or fusion proteins, wherein a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain of the antibody, fragment or fusion protein comprise specific amino acid substitutions as disclosed herein. The specific amino acid substitutions result in a preferential pairing of the CH1 and CL domains with the specific amino acid substitutions such that mispairing with one or more constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain of a different amino acid sequence can be strongly reduced, if not avoided. The present invention is in particular relevant for at least one nucleic acid encoding a multispecific antibody or fragment thereof or fusion protein or a mixture of antibodies or fragments thereof or fusion proteins, wherein the at least one nucleic acid may be administered to a patient to have the encoded antibody or fragment thereof or fusion protein produced by the patient's cells. The present invention is also concerned with in vitro methods of determining correct pairing between heavy and light chains.

Description

Our ref.: B19030WO / LEENGINEERED CH1 AND CL DOMAINS FOR THE PREVENTION OF CHAIN MISPAIRINGTECHNICAL FIELD

[0001] The present invention is in the field of antibodies or fragments thereof as well as fusion proteins comprising the same, and nucleic acids encoding the afore-mentioned antibodies or fragments thereof or fusion proteins, wherein a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain of the antibody, fragment or fusion protein comprise specific amino acid substitutions as disclosed herein. Thus, the present invention is concerned with at least one nucleic acid encoding at least a CH1 domain and a CL domain, wherein the CH1 domain and the CL domain comprise specific amino acid substitutions as disclosed herein. The present invention is also concerned with a composition comprising the at least one nucleic acid and the use of such a composition in therapy. Further, the present invention relates to an antibody or fragment thereof comprising a CH1 domain and a CL domain, wherein each domain comprises specific amino acid substitutions as disclosed herein. Corresponding fusion proteins are also part of the present invention. Further, the present invention relates to mixtures of at least two antibodies and / or fragments and / or fusion proteins, wherein a first antibody or fragment thereof or fusion protein comprises a CH1 and a CL domain, wherein each domain comprises specific amino acid substitutions as disclosed herein; and each further antibody or fragment thereof or fusion protein does not comprise such CH1 and CL domains. The present invention is also concerned with a composition comprising the antibody, fragment or fusion protein and the use of such a composition in therapy or in diagnosis. The present invention is also concerned with an in vitro method of determining the percent of correctly paired heavy and light chains amongst paired antibody chains of at least two different antibodies; and an in vitro method of detecting mispaired heavy and light chains amongst paired chains of an IgG antibody and of a Fab fragment.BACKGROUND OF THE INVENTION

[0002] Antibodies are essential components of the immune system and are typically composed of two heavy chains and two light chains that form a Y-shaped structure. The correct pairing of the constant heavy chain domain CH1 and the constant light chain domain CL is, amongst other factors, crucial for the stability and functionality of antibodies.

[0003] During the production of multispecific (in particular bispecific) antibodies or fragments thereof, or in case of mixtures of two or more antibodies or fragments thereof with different specificities, the different CH1 and CL domains can mispair, which often leads to the formation of non-functional or unstable antibodies or fragments thereof, which can impact the efficacy and safety of antibody-based therapeutics. Therefore, it is essential to ensure proper pairing of CH1 and CL domains during the production of antibodies or fragments thereof, in particular of bispecific antibodies or fragmentsLE:LEthereof, or if two or more antibodies or fragments thereof with different specificities are produced and subsequently present in a mixture.

[0004] While approaches providing proper CH1 and CL pairing are useful and have been provided for classical protein antibody therapeutics (see e.g. WO 2015 / 150447), such approaches are especially useful for nucleic acid-encoded (in particular mRNA-encoded) antibodies or fragments thereof, a new class of biological therapeutics that, unlike traditional antibody-based therapies, are produced within the patient's body. Since the antibodies or fragments thereof are produced in situ after nucleic acid (in particular mRNA) delivery to the patient's cells, approaches to reduce mispairing based on purifications or the like are not feasible.

[0005] Accordingly, a correct pairing of CH1 and CL is of utmost importance for drug safety and efficacy of nucleic-acid encoded antibodies or fragments thereof, and there is the need to provide approaches ensuring a correct pairing. Such approaches can of course also be used for the antibodies or fragments thereof as such, i.e. they are not limited to nucleic acid encoded antibodies or fragments thereof. Furthermore, such approaches can also be useful for fragments comprising or consisting of corresponding CH1-CL domains, which can, if desired, easily be fused to at least one peptide.SUMMARY OF THE INVENTION

[0006] The present application inter alia provides an approach of substituting specific amino acids in the CH1 domain and the corresponding CL domain such that the resulting CH1 domain and the resulting CL domain preferentially bind to each other. The corresponding amino acids may also be referred to as complementarily matched amino acids within the CH1 and CL domains and are assumed to be located within or at least influencing residues within the interaction interface of the CH1 and CL domains. If a nucleic acid, in particular an mRNA, encoding such CH1 and CL domains is administered to a patient such that the nucleic acid is expressed by cells of the patient in order to produce an antibody or a fragment thereof comprising such CH1 and CL domains, or a fusion protein comprising the antibody or fragment thereof, the mispairing of such CH1 and CL domains with additionally present CH1 and CL1 domains (not comprising these amino acid substitutions) is greatly reduced, thus resulting in a higher number of correctly paired antibodies or fragments thereof or fusion proteins.

[0007] In a first aspect, the present application is directed to at least one nucleic acid encoding at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU; preferably (1 a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU, and (1 a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214Vin the numbering according to EU; or (1 b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU, and (1 b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU;in the numbering according to EU;(2)(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141 Y, L145D and K147S in the numbering according to EU, and (2)(ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU;(3)(i) a CH1 domain comprising the amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU, and (3)(ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU;(4)(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU, and (4)(ii) a CL domain comprising the amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU;(5)(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141 W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU, and (5)(ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU;(6)(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU, and (6)(ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU;(7)(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU, and (7)(ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU;(8)(i) a CH1 domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU, and (8)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numberingaccording to Kabat corresponding to S162C and C214V in the numbering according to EU; preferably (8a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU, and (8a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU;(9)(i) a CH1 domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or (i”) G141W and K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’") A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU, and (9)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU; and(10)(i) a CH1 domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (10)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU.

[0008] In an embodiment of the first aspect, the at least one nucleic acid encodes at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat; preferably (1a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat, and (1 a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat; or (1 b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat, and (1 b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat;(2)(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat, and (2)(ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat;(3)(i) a CH1 domain comprising the amino acid substitutions S130M, G141 W and H172D in the numbering according to Kabat, and (3)(ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat;(4)(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat, and (4)(ii) a CL domain comprising the amino acid substitutions F118G and S176F in the numbering according to Kabat;(5)(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141Wand K221 E in the numbering according to Kabat corresponding or (i”) L124I, G141 F and K221 D in the numbering according to Kabat, and (5)(ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat;(6)(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat, and (6)(ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat;(7)(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat, and (7)(ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat;(8)(i) a CH1 domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat, and (8)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat, preferably (8a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat, and (8a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat;(9)(i) a CH1 domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat or (i”) G141 W and K222R in the numbering according to Kabat or (i’”) A125M, S127I and V190W in the numbering according to Kabat, and (9)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat; and(10)(i) a CH1 domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat, and (10)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat.

[0009] In an embodiment of the first aspect, the at least one nucleic acid encodes at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S136C and C220V in the numbering according to EU, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to EU, preferably (1 a)(i) a CH1 domain comprising the amino acid substitutions S136C, L145D, K147S and C220V in the numbering according to EU, and (1 a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to EU; or (1 b)(i) a CH1 domain comprising the amino acid substitutions S136C, H168D and C220V in the numbering according to EU, and (1 b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to EU;(2)(i) a CH1 domain comprising the amino acid substitutions A141Y, L145D and K147S in the numbering according to EU, and (2)(ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to EU;(3)(i) a CH1 domain comprising the amino acid substitutions S134M, G143W and H168D in the numbering according to EU, and (3)(ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to EU;(4)(i) a CH1 domain comprising the amino acid substitutions L128I, G143W, F170S and S183A in the numbering according to EU, and (4)(ii) a CL domain comprising the amino acid substitutions F118G and S176F in the numbering according to EU;(5)(i) a CH1 domain comprising the amino acid substitutions (i’) L128I, G143W, and K213E in the numbering according to EU or (i”) L128I, G143F and K213D in the numbering according to EU; and (5)(ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to EU;(6)(i) a CH1 domain comprising the amino acid substitutions S134M, L145D, K147S andV185Y in the numbering according to EU, and (6)(ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to EU;(7)(i) a CH1 domain comprising the amino acid substitutions K147S, F170S, S181 E andS183A in the numbering according to EU, and (7)(ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to EU;(8)(i) a CH1 domain comprising the amino acid substitutions F170C and C220V in the numbering according to EU, and (8)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numbering according to EU, preferably (8a)(i) a CH1 domain comprising the amino acid substitutions L145D, K147S, F170C and C220V in the numbering according to EU, and (8a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to EU;(9)(i) a CH1 domain comprising the amino acid substitutions (i’) T139W, T169V and V185W in the numbering according to EU or (ii’) G143W and K214R in the numbering according to EU or (iii’) A129M, S131 I and V185W in the numbering according to EU, and (9)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to EU; and(10)(i) a CH1 domain comprising the amino acid substitutions H168Y and K213E in the numbering according to EU, and (10)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to EU.

[0010] In an embodiment of the first aspect, the at least one nucleic acid is one (i.e. a single) nucleic acid that encodes at least the CH1 domain and the CL domain.

[0011] In a preferred embodiment of the first aspect, the at least one nucleic acid corresponds to two nucleic acids, wherein the first nucleic acid encodes at least the CH1 domain and the second nucleic acid encodes at least the CL domain. Thus, if e.g. an antibody consisting of complete heavy and light chains is encoded, the first nucleic acid may encode the heavy chain of the antibody (comprising the CH1 domain) and the second nucleic acid may encode the light chain of the antibody (comprising the CL domain). If e.g. a fragment thereof is encoded, the first nucleic acid may encode the fragment of the heavy chain (comprising or consisting of the CH 1 domain) and the second nucleic acid may encode the full light chain or a fragment of the light chain (comprising or consisting of the CL domain). The same applies to at least one nucleic acid encoding a fusion protein of the antibody or fragment thereof,wherein in addition to the antibody or fragment thereof a peptide is encoded that is fused to the N- or C-terminus of the corresponding heavy and light chains (or fragments thereof). This is set out further in the next embodiments.

[0012] In an embodiment of the first aspect, the at least one nucleic acid encodes an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the CH1 domain and the CL domain. The at least one nucleic acid may be one nucleic acid that encodes the antibody or fragment thereof. Preferably, the at least one nucleic acid may be two nucleic acids, wherein the first nucleic acid encodes a heavy chain comprising the CH1 domain, or a fragment thereof comprising or consisting of the CH1 domain, and the second nucleic acid encodes a light chain comprising the CL domain, or a fragment thereof comprising or consisting of the CL domain.

[0013] In an embodiment of the first aspect, the at least one nucleic acid encodes a fusion protein comprising an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the CH1 domain and the CL domain. The at least one nucleic acid may be one nucleic acid that encodes the fusion protein, wherein the antibody or fragment thereof is N- and / or C-terminally fused to a peptide (e.g. a cytokine, a receptor, a ligand or a binding fragment). Alternatively, the at least one nucleic acid may be two nucleic acids, wherein the first nucleic acid encodes a heavy chain comprising the CH1 domain, or a fragment thereof comprising or consisting of the CH1 domain, which is N- and / or C- terminally fused to a peptide (e.g. a cytokine, a receptor, a ligand or a binding fragment) and the second nucleic acid encodes a light chain comprising the CL domain, ora fragment thereof comprising or consisting of the CL domain. Yet alternatively, the at least one nucleic acid may be two nucleic acids, wherein the first nucleic acid encodes a heavy chain comprising the CH1 domain, or a fragment thereof comprising or consisting of the CH1 domain, and the second nucleic acid encodes a light chain comprising the CL domain, or a fragment thereof comprising or consisting of the CL domain, which is N- and / or C-terminally fused to a peptide (e.g. a cytokine, a receptor, a ligand or a binding fragment). Yet alternatively, the at least one nucleic acid may be two nucleic acids, wherein the first nucleic acid encodes a heavy chain comprising the CH1 domain, or a fragment thereof comprising or consisting of the CH1 domain, which is N- and / or C-terminally fused to a peptide (e.g. a cytokine, a receptor, a ligand or a binding fragment), and the second nucleic acid encodes a light chain comprising the CL domain, or a fragment thereof comprising or consisting of the CL domain, which is N- and / or C- terminally fused to a peptide (e.g. a cytokine, a receptor, a ligand or a binding fragment).

[0014] In an embodiment of the first aspect, the at least one nucleic acid encodes an IgG antibody or IgG antibody fragment thereof.

[0015] In an embodiment of the first aspect, the at least one nucleic acid encodes an lgG1 antibody or IgG 1 antibody fragment thereof.

[0016] In an embodiment of the first aspect, the at least one nucleic acid encodes a CL domain that is of kappa isotype.

[0017] In an embodiment of the first aspect, the at least one nucleic acid encodes an antibody selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.

[0018] In an embodiment of the first aspect, the at least one nucleic acid encodes a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment. It is notedthat the CH1-CL fragment is the smallest fragment that is encoded, which can in particular be interesting in terms of encoding fusion proteins that comprise such a CH1-CL fragment, as discussed next.

[0019] In an embodiment of the first aspect, the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof. Because the antibody or fragment thereof is comprised of at least two chains or fragments thereof, there are in principle one N- and one C-terminus per chain such that a peptide may in principle be fused to the antibody or fragment thereof at the at least two N-termini, at the at least two C-termini or at all termini, i.e. the two N-termini and two C-termini. If the antibody or fragment thereof is comprised of four chains or fragments thereof (as is e.g. the case for an IgG antibody), there are in principle eight termini, to which a peptide may be fused. However, depending on the fusion protein of interest, the peptide may also be fused to the antibody or fragment thereof at one, two, three or four of the four termini or one, two, three, four, five, six, seven or eight of the eight termini. In one embodiment of the fusions proteins comprising an IgG or Fab antibody or fragment, the peptide(s) is / are preferably fused to the C terminus.

[0020] In an embodiment of the first aspect, the encoded fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody-peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2- peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptidei-2 (preferably CH1-CL- peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2).

[0021] In an embodiment of the first aspect, the peptide (of the encoded fusion protein) is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH. However, it is emphasized that there is no limitation as regards the peptide to be fused.

[0022] In an embodiment of the first aspect, the encoded fusion protein is selected from the group consisting of symmetric IgG antibody-cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab-cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2- SCFV2, F(ab’)2-VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL- scFv, CH1-CL-VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL- scFv, SCFV2-CH 1 -CL-SCFV2, VHH2-CH1-CL-VHH2, SCFV2-CH 1 -CL-VHH2and VHH2-CH 1 -CL-SCFV2.Embodiments relating to encoded antibodies or fragments thereof or fusion proteins comprising at least two different CH1 domains and at least two different CL domains

[0023] The present embodiments in particular relate to an encoded multispecific antibody or fragment thereof or a fusion protein comprising the antibody or fragment thereof, which comprises at least two different CH1 domains and at least two different CL domains.

[0024] It can be preferred that the encoded multispecific antibody or fragment thereof or fusion protein comprising the antibody or fragment thereof is a bispecific antibody or fragment thereof, which comprises two different CH1 domains and two different CL domains, wherein a first CH1 domain anda first CL domain are according to the present invention and the further CH1 domains and CL domains are different therefrom (as set out in detail below). Because the encoded variable domains VH and VL, which are present N-terminally of the CH and CL domains, are different for the different CH1 / CL domains, the resulting antibody or fragment thereof or fusion protein comprising the antibody or fragment thereof is at least bispecific. As an example, the at least one nucleic acid may encode an asymmetric IgG antibody with two different sets of VH and VL domains (thus resulting in the antibody being bispecific), wherein the amino acid substitutions in the CH1 and CL domains of the present invention are present in “one arm”, i.e. present only in one of the two light chains and in one of the two heavy chains, and present such that they result not only in preferential binding of the corresponding CH1 and CL domains but also in the correct pairing of the VH and VL domains (creating one of the two desired antigen binding regions). The “other arm” may e.g. comprise wild-type CH1 and CL domains in the corresponding heavy and light chains, which will correspondingly assemble and result in the correct pairing of the further two VH and VL domains (creating the second of the two desired antigen binding regions).

[0025] In an embodiment of the first aspect, the at least one nucleic acid encodes an antibody or fragment thereof, or a fusion protein comprising the antibody or fragment thereof, wherein the antibody or fragment thereof comprises at least two different CH1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are selected from the group consisting of (1)(i) and (1)(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each additional CH1 domain comprises a CH1 domain different from (1 )(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and each additional CL domain comprises a CL domain different from (1)(ii), (2)(ii), (3)(ii), (4)(ii), (5)(ii), (6)(ii), (7)(ii), (8)(ii), (9)(ii), and (10)(ii).

[0026] In an embodiment of the first aspect, each additional CH1 domain matches an additional CL domain.

[0027] In an embodiment of the first aspect, an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain.

[0028] In an embodiment of the first aspect, the encoded antibody or fragment thereof is an IgG antibody or IgG antibody fragment thereof.

[0029] In an embodiment of the first aspect, the encoded antibody or fragment thereof is an IgG 1 antibody or IgG 1 antibody fragment thereof.

[0030] In an embodiment of the first aspect, the CL domain of the encoded antibody or fragment thereof is of kappa isotype.

[0031] In an embodiment of the first aspect, the encoded antibody or fragment thereof is selected from the group consisting of a symmetric or asymmetric IgG antibody and a F(ab’)2 fragment. Such antibodies or fragments are preferred because they comprise two sets of CH1 and CL domains, which may differ as set out above. However, the encoded antibody or fragment thereof is not limited thereto but any other antibody or fragment thereof comprising at least two CH1 and CL domains can be encoded by the at least one nucleic acid.

[0032] In an embodiment of the first aspect, the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof. Because the antibody or fragment thereof is comprised of at least two chains or fragments thereof, there are in principle one N- and one C-terminus per chain such that a peptide may in principle be fused to the antibody or fragment thereof at the at least two N-termini, at the at least two C-termini or at all termini, i.e. the at least two N-termini and at least two C-termini. If the antibody or fragment thereof is comprised of four chains or fragments thereof (as is e.g. the case for an IgG antibody), there are in principle eight termini, to which a peptide may be fused. However, depending on the fusion protein of interest, the peptide may also be fused to the antibody or fragment thereof at one, two, three or four of the four termini or one, two, three, four, five, six, seven or eight of the eight termini. In one embodiment of the fusions proteins comprising an IgG or Fab antibody or fragment, the peptide(s) is / are preferably fused to the C terminus.

[0033] In an embodiment of the first aspect, the encoded fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody-peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2- peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptidei-2 (preferably CH1-CL- peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2).

[0034] In an embodiment of the first aspect, the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv, Fab and VHH. However, it is emphasized that there is no limitation as regards the peptide to be fused.

[0035] In an embodiment of the first aspect, the encoded fusion protein is selected from the group consisting of a symmetric IgG antibody-cytokine-i-8 (preferably symmetric IgG antibody-cytokine2), asymmetric IgG antibody-cytokine-i-8 (preferably asymmetric IgG antibody-cytokine2), asymmetric IgG antibody-scFvi-s (preferably asymmetric IgG antibody-scFv2), asymmetric IgG antibody-VHHi-s (preferably asymmetric IgG antibody-VHH2), asymmetric IgG antibody-Fabi-s (preferably IgG antibody-Fab), Fab-scFvi-4 (preferably Fab-scFv2), Fab-VHHi-4 (preferably Fab-VHH2), F(ab’)2-scFvi- 8 (preferably F(ab’)2-scFv2), and F(ab’)2-VHHi-8 (preferably F(ab’)2-VHH2), asymmetric IgG antibody- Fabi-s (preferably asymmetric IgG antibody-Fab), Fab-scFvi-4 (preferably Fab-scFv2), or Fab-VHHi-4 (preferably Fab-VHH2).

[0036] In an embodiment of the first aspect, the encoded antibody or fragment thereof or fusion protein comprises two different CH1 domains and two different CL domains. In this embodiment, the encoded antibody or fragment thereof or fusion protein is preferably bispecific. However, because of the option of fusing further antigen-binding domains (e.g. scFv or VHH), a resulting encoded fusion protein may also be multispecific.Embodiments relating to at least two encoded antibodies or fragments thereof or fusion proteins

[0037] The present embodiments in particular relate to at least two encoded antibodies or fragments thereof or fusion proteins comprising the antibody or fragment thereof, wherein it can be preferred thateach of the encoded antibodies or fragments thereof or fusion proteins is monospecific. Each CH1 domain and CL domain of each encoded antibody or fragment thereof or fusion protein may thus be identical, contrary to the embodiments above relating to a multispecific antibody or fragment thereof or fusion protein. Because the encoded variable domains VH and VL, which are present N-terminally of the CH and CL domains, are different for the different CH1 / CL domains, the specificities of the encoded at least two antibodies or fragments thereof or fusion proteins are different. The present embodiments are in particular relevant for at least one nucleic acid encoding e.g. two antibodies, wherein the at least one nucleic acid is administered to a patient. Upon administration, the patient’s cells will produce the encoded two antibodies and it is crucial that these two antibodies are assembled correctly in terms of light chain-heavy chain assembly because if the light chain of the first antibody would assemble with the heavy chain of the second antibody, the wrong VH and VL domains would be assembled such that there would be no desired antigen-binding.

[0038] In an embodiment of the first aspect, the at least one nucleic acid encodes at least two antibodies or fragments thereof, or fusion proteins comprising the antibody or fragment thereof, wherein each CH1 domain and each CL domain of an encoded first antibody or fragment thereof or fusion protein is selected from the group consisting of (1)(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each CH1 domain and each CL domain of each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain different from (1 )(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and a CL domain different from (1)(ii), (2)(ii), (3)(ii), (4)(ii), (5)(ii), (6)(ii), (7)(ii), (8)(ii), (9)(ii), and (10)(ii).

[0039] In an embodiment of the first aspect, each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additionally encoded antibody or fragment thereof or fusion protein.

[0040] In an embodiment of the first aspect, an additionally encoded antibody or fragment thereof comprises a wild-type CH1 domain and a wild-type CL domain.

[0041] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are antibodies, preferably two antibodies. In another embodiment, the at least two encoded antibodies or fragments thereof or fusion proteins are fragments, preferably two fragments. In another embodiment, the at least two encoded antibodies or fragments thereof or fusion proteins are an antibody and a fragment, preferably one antibody and one fragment. In another embodiment, the at least two encoded antibodies or fragments thereof or fusion proteins are fusion proteins, preferably two fusion proteins. In another embodiment, the at least two encoded antibodies or fragments thereof or fusion proteins are an antibody or fragment thereof and a fusion protein, preferably one antibody or fragment thereof and one fusion protein.

[0042] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise IgG antibodies or IgG antibody fragments.

[0043] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise IgG 1 antibodies or IgG 1 antibody fragments.

[0044] In an embodiment of the first aspect, the CL domains of the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise CL domains of kappa isotype.

[0045] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise antibodies selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.

[0046] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.

[0047] In an embodiment of the first aspect, the at least two encoded antibodies or fragments thereof or fusion proteins are or comprise a fusion protein, wherein a peptide is fused to the N- and / or C- terminus of the antibody or fragment thereof. Because the antibody or fragment thereof is comprised of at least two chains or fragments thereof, there are in principle one N- and one C-terminus per chain such that a peptide may in principle be fused to the antibody or fragment thereof at the at least two N- termini, at the at least two C-termini or at all termini, i.e. the two N-termini and two C-termini. If the antibody or fragment thereof is comprised of four chains or fragments thereof (as is e.g. the case for an IgG antibody), there are in principle eight termini, to which a peptide may be fused. However, depending on the fusion protein of interest, the peptide may also be fused to the antibody or fragment thereof at one, two, three or four of the four termini or one, two, three, four, five, six, seven or eight of the eight termini. In one embodiment of the fusions proteins comprising an IgG or Fab antibody or fragment, the peptide(s) is / are preferably fused the C terminus(i).

[0048] In an embodiment of the first aspect, the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody-peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody- peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptidei-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2).

[0049] In an embodiment of the first aspect, the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

[0050] In an embodiment of the first aspect, the fusion protein is selected from the group consisting of symmetric IgG antibody-cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab-cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2- VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1-CL- VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2-CH1- CL-SCFV2, VHH2-CHI-CL-VHH2, SCFV2-CH 1 -CL-VHH2and VHH2-CH1-CL-scFv2.Further general embodiments

[0051] In an embodiment of the first aspect, at least a CH1 domain is encoded by a first nucleic acid and at least a CL domain is encoded by a second nucleic acid.

[0052] In an embodiment of the first aspect, the encoded antibody or fragment thereof or fusion protein comprises two CH3 domains, wherein the two CH3 domains comprise amino acid substitutions in the CH3 domains known as 'knobs-into-holes'. Such CH3 substitutions aim at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. Thus, in one chain bulky amino acids may be replaced by amino acids with short side chains to create a 'hole'. Conversely, amino acids with large side chains may be introduced into the other CH3 domain, to create a 'knob'. By coexpressing these two heavy chains, high yields of heterodimer formation ('knob-hole') versus homodimer formation ('hole-hole' or 'knob-knob') was observed (see e.g. WO 96 / 027011). The percentage of heterodimer may be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681 ; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). Such CH3 domain substitutions are in particular relevant for the present invention if the at least one nucleic acid encodes at least two antibodies or fragments thereof or fusion proteins, where not only the correct pairing between the CH 1 and CL domains is crucial but also the correct pairing between the two heavy chains (via CH3 domains). There exist several approaches for CH3-modifications to enforce the heterodimerization, which are described e.g. in WO 96 / 027011 , WO 98 / 050431 , EP 1870459, WO 2007 / 110205, WO 2007 / 147901 , WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291 . Typically, in all such approaches the first CH3 domain and the second CH3 domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) cannot longer homodimerize with itself but is forced to heterodimerize with the complementary engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). These different approaches for improved heavy chain heterodimerization are contemplated in combination with the introduction of substitutions as disclosed herein in the CH1 and CL domains in the encoded antibodies or fragments thereof or fusion proteins, where the correct pairing between heavy chains or fragments thereof is critical.

[0053] In an embodiment of the first aspect, the nucleic acid is DNA or RNA, preferably mRNA.

[0054] In an embodiment of the first aspect, the coding sequence of the nucleic acid is codon- optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content does not change the sequence of the encoded amino acid sequence. Alternatively, the G / C content of the codon-optimized nucleic acid is decreased compared to wild type coding sequence, wherein the codon-optimization and / or the decrease in the G / C content does not change the sequence of the encoded amino acid sequence.

[0055] In an embodiment of the first aspect, the at least one nucleic acid is RNA.Embodiments relating to the nucleic acid being at least one RNA, preferably mRNA

[0056] Generally, the nucleic acid being an RNA may be equipped with structural elements enabling or improving translation and or immunogenicity. Thus, the RNA may comprise elements selected fromthe group consisting of a cap structure, a 5’ UTR, 3’ UTR, poly A tail and one or more modified nucleotides replacing any of adenosine, cytidine, guanosin and / or uridine in the RNA.

[0057] In an embodiment, the RNA comprises a modified nucleoside in place of uridine. In such a case there is preferably a modified nucleoside in place of each or essentially each uridine in the RNA. Preferably, the modified nucleoside is selected from pseudouridine (i ), N1-methyl-pseudouridine (m1 qj), and 5-methyl-uridine (m5U).

[0058] In an embodiment, the RNA comprises the 5’ cap m27’3'oGppp(mi2'o)ApG.

[0059] In an embodiment, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 51 , or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 51 .

[0060] In an embodiment, the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 52, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 52.

[0061] In an embodiment, the RNA comprises a poly-A sequence. Preferably, the poly-A sequence comprises at least 100 nucleotides. The poly-A sequence may comprise or consist of the nucleotide sequences of SEQ ID NO:53.

[0062] In an embodiment,(i) the RNA comprises a modified nucleoside in place of each uridine; and / or(ii) the RNA comprises a modified nucleoside in place of each uridine, wherein the modified nucleoside is independently selected from pseudouridine (i ), N1-methyl-pseudouridine (ml i ), and 5- methyl-uridine (m5U); and / or(iii) the RNA comprises the 5’ cap m27’3'oGppp(mi2'o)ApG; and / or(iv) the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 51 , or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 51 ; and / or(v) the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 52, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 52; and / or(vi) the RNA comprises a poly-A tail comprising the nucleotide sequence of SEQ ID NO: 53.

[0063] In an embodiment,(i) the RNA comprises a modified nucleoside in place of each uridine;(ii) the RNA comprises a modified nucleoside in place of each uridine, wherein the modified nucleoside is independently selected from pseudouridine (i ), N1-methyl-pseudouridine (ml i ), and 5- methyl-uridine (m5U);(iii) the RNA comprises the 5’ cap m27’3'oGppp(mi2'o)ApG;(iv) the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 51 , or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 51 ;(v) the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 52, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 52; and(vi) the RNA comprises a poly-A tail comprising the nucleotide sequence of SEQ ID NO: 53.

[0064] In a second aspect, the present application is directed to a particle comprising the at least one nucleic acid according to the first aspect, including all embodiments of the first aspect.

[0065] In one embodiment of the second aspect, the particle is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a baculoviral vector, an Epstein-Barr viral vector, a poxvirus vector, a virosome, a lipid nanoparticle (LNP), a liposome, a lipoplex (LPX), and a polyplex (PLX).

[0066] In one embodiment of the second aspect, the at least one nucleic acid is fully or partially encapsulated within an LNP, liposome, LPX or PLX.

[0067] In one embodiment of the second aspect, the at least one nucleic acid is encapsulated in an LNP.

[0068] In a third aspect, the present application is directed to a composition comprising the at least one nucleic acid according to the first aspect, including all embodiments of the first aspect, or the particle according to the second aspect, including all embodiments of the second aspect.

[0069] In an embodiment of the third aspect, the composition is a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.

[0070] In a fourth aspect, the present application is directed to the composition of the third aspect for use in therapy.

[0071] In a fifth aspect, the present application is directed to the composition of the third aspect for use in the treatment of cancer.

[0072] In a sixth aspect, the present application is directed to the composition of the third aspect for use in the treatment of an infectious disease.

[0073] In a seventh aspect, the present application is directed to the composition of the third aspect for use in the treatment of an autoimmune disease.

[0074] In an eighth aspect, the present application is directed to an in vitro method of determining the percent of correctly paired heavy and light chains amongst paired heavy and light chains derived from at least two antibodies directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising at least (i) a heavy chain and a light chain of a first antibody and (ii) a light chain of a second antibody; b) determining the total amount of antibody by staining the paired heavy and light chains with an anti-Fc antibody and quantifying the signal of the anti-Fc antibody to arrive at a quantity 1 ; c) determining the amount of the first antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the first antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the first antibody,and quantifying the signal of said anti-idiotype antibody to arrive at a quantity 2a; d) calculating a result according to the formula [quantity 2a I quantity 1] x 100; wherein the result indicates the percent of correctly paired heavy and light chains.

[0075] The term “derived from at least two antibodies” as used in the eighth aspect does not imply that all chains of the at least two antibodies are present. Rather, step a) defines which chains of which antibody are comprised in the provided paired heavy and light chains.

[0076] In one embodiment of the eighth aspect, (i) the light chain and a heavy chain of the second antibody are provided in step a); (ii) the method comprises an additional step c1) of determining the amount of the second antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the second antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the second antibody, and quantifying the signal of said antiidiotype antibody to arrive at a quantity 2b; and (iii) step d) comprises calculating a result according to the formula [(quantity 2a + quantity 2b) I quantity 1] x 100.

[0077] In one embodiment of the eighth aspect, the at least two antibodies directed to different epitopes are IgG antibodies.

[0078] In one embodiment of the eighth aspect, the at least two antibodies directed to different epitopes are two antibodies.

[0079] In one embodiment of the eighth aspect, the at least two antibodies are more than two antibodies, e.g. 3, 4, 5 or 6 antibodies, wherein each antibody is preferably directed to a different epitope. In this embodiment, it is understood that there is not only step c) (and optionally step c1) as outlined in an embodiment above) relating to the quantification of the first (and optionally of the second antibody as outlined in an embodiment above) but that there are further quantification steps for the remaining antibodies using for each additional antibody an anti-idiotype antibody, wherein said antiidiotype antibody binds only the correctly assembled chains of the respective additional antibody. Further, it is understood in this embodiment that step d) comprises the calculation of a result according to the formula [(quantity 2a + quantity 2b + all quantities obtained for an antibody / antibodies in addition to two antibodies) I quantity 1] x 100.

[0080] In one embodiment of the eighth aspect, the paired heavy and light chains are provided in step a) by contacting the heavy chain and the light chain of a first antibody (wherein these two chains are preferably provided in a non-paired manner) with at least the light chain of a second antibody under conditions that allow for the pairing of these chains and the formation of disulfide bonds, Thus, the chains, i.e., the proteins, may be contacted with each other under the afore-mentioned conditions to arrive at paired heavy and light chains.

[0081] In one embodiment of the eighth aspect, the paired heavy and light chains are provided in step a) by expressing at least one nucleic acid encoding at least the heavy and the light chain of the first antibody and the light chain of the second antibody in a cell (optionally resulting in the pairing of the chains and the formation of disulfide bonds) to obtain paired heavy and light chains. In this embodiment, it is understood that the at least one nucleic acid encodes the chains of all antibodies if more than two antibodies are concerned, e.g. the chains of 3, 4, 5 or 6 antibodies. In this embodiment, the paired heavy and light chains may be comprised in a cell culture supernatant. The paired heavyand light chains may be purified from the cell culture supernatant, optionally to remove further components comprised in the cell culture supernatant and further optionally wherein the paired chains are comprised in a solution after the purification. It is understood that such a purification, if carried out, does not impact the pairing of the chains, i.e. the paired heavy and light chains remain paired heavy and light chains during the purification and corresponding conditions are chosen. Further, the heavy and light chains may be concentrated but still be comprised in cell culture supernatant or the paired heavy and light chains may be concentrated after the purification, optionally wherein the paired heavy and light chains are comprised in a solution after the concentration. It is understood that such a concentration, if carried out, does not impact the pairing of the chains, i.e. the paired heavy and light chains remain paired heavy and light chains during the concentration and corresponding conditions are chosen. In this embodiment, the at least one nucleic acid is expressed by transfecting the cell with the afore-mentioned at least one nucleic acid, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the at least one nucleic acid is expressed transiently and / or stably. In other words, a transient expression of the encoded heavy and light chains in the cells or a stable expression of the encoded heavy and light chains in the cells can be used when it comes to the expression. A stable expression may in particular be achieved by integrating corresponding expression cassettes into the genome of the cell. Furthermore, inducible expression systems may be used to be able to switch the expression on and off.

[0082] In one embodiment of the eighth aspect, the paired heavy and light chains are comprised in a solution.

[0083] In one embodiment of the eighth aspect, the determination steps b), c) and c1) are carried out using an enzyme-linked immunosorbent assay (ELISA). Any suitable ELISA may be used, including a Gyros ELISA. A sandwich ELISA is particularly preferred for the present method.

[0084] In one embodiment of the eighth aspect, the determination step b) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety- coupled anti-human IgG-Fcy antibody as detection agent. The anchoring moiety is preferably biotin such that the capture reagent is preferably a biotinylated anti-human IgG-Fc antibody. The reporter moiety is preferably a fluorophore such that the detection agent is preferably a fluorophore-coupled anti-human IgG-Fcy antibody.

[0085] In one embodiment of the eighth aspect, the determination step c) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety- coupled anti-idiotype antibody directed to the first antibody as detection agent. The anchoring moiety is preferably biotin such that the capture reagent is preferably a biotinylated anti-human IgG-Fc antibody. The reporter moiety is preferably a fluorophore such that the detection agent is preferably a fluorophore-coupled anti-idiotype antibody directed to the first antibody.

[0086] In one embodiment of the eighth aspect, the determination step c1) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety- coupled anti-idiotype antibody directed to the second antibody as detection agent. The anchoring moiety is preferably biotin such that the capture reagent is preferably a biotinylated anti-human IgG-Fc antibody. The reporter moiety is preferably a fluorophore such that the detection agent is preferably a fluorophore-coupled anti-idiotype antibody directed to the second antibody.

[0087] It is understood that in the eighth aspect that further determination steps, if carried out because the at least two antibodies are more than two antibodies, e.g. 3, 4, 5 or 6 antibodies, the corresponding determination steps may be carried out by using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety-coupled anti-idiotype antibody directed to each additional antibody. The anchoring moiety is preferably biotin such that the capture reagent is preferably a biotinylated anti-human IgG-Fc antibody. The reporter moiety is preferably a fluorophore such that the detection agent is preferably a fluorophore-coupled anti-idiotype antibody directed to each of the additional antibodies, wherein the reporter moiety (and preferably the fluorophore) differ in these embodiments for each additional antibody.

[0088] In one embodiment of the eighth aspect, the result of step d) is compared to a standard curve to account for not assembled chains (which are not detected by the at least one anti-idiotype antibody), wherein the standard curve has been obtained by determining in samples of purified antibody mixtures with different, pre-established and thus known percentages of mispairing, the percent of correctly paired heavy and light chains, preferably mispaired antibodies, and wherein the standard curve indicates the difference between the pre-established and thus known percentage of correctly paired heavy and light chains in the samples of purified antibody mixtures vs. the determined percentage of correctly paired heavy and light chains in these samples. It is understood that the different, known percentages of mispairing are preferably increasing percentages of mispairing covering the relevant percentages of mispairing. Using such a standard curve, the difference at the determined percentage, i.e., the result of step d), to the known percentage may be taken into account by multiplying the result of step d) with the factor (indicating the deviation) obtained from the standard curve. An exemplary standard curve and how such a standard curve has been obtained is disclosed herein in particular in Example 3.3 and Figure 8.

[0089] In a ninth aspect, the present application is directed to an in vitro method of detecting mispaired heavy and light chains amongst paired heavy and light chains derived from at least one IgG antibody and at least one Fab fragment directed to different epitopes (i.e., the at least one IgG antibody and the at least one Fab fragment are directed to different epitopes), wherein the method comprises the following steps: a) providing paired heavy and light chains comprising (i) a heavy chain and a light chain of an IgG antibody, wherein the heavy chain is not fused to a tag and the light chain is fused to a tag1 , and (ii) a VH-CH1 -fusion chain and a light chain of a Fab fragment, wherein the VH-CH1- fusion chain is not fused to a tag and the light chain is fused to a tag2; b) denaturing the paired heavy and light chains provided in step a) under non-reducing conditions; c) separating the denatured paired heavy and light chains according to the molecularweight; and d) staining the paired heavy and light chains separated according to molecular weight with (i) a detection agent specific for tag1 , (ii) a detection agent specific for tag2; and optionally (iii) adetection agent specific for human Fd; wherein the staining of the different detection agents can be discriminated from each other; wherein tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is such that a discrimination between tag1 and tag2 is possible when separated according to the molecular weight; and wherein mispaired heavy and light chains are identified via their molecular weight and stain; and optionally wherein the ratio of (i) mispaired heavy and light chains to (ii) correctly paired heavy and light chains and single chains is identified via the Fd staining.

[0090] In one embodiment of the ninth aspect, (i) paired heavy and light chains of a molecular weight of about (50 kDa + MW1) stained with the agent specific for tag1 ; (ii) paired heavy and light chains of about (75 kDa + MW2) stained with the agent specific for tag2; (iii) paired heavy and light chains of about (150 kDa + MW1 + MW2) stained with the agent specific for tag 1 and the agent specific for tag2; and (iv) paired heavy and light chains of about (150 kDa + M W2 + M W2) stained with the agent specific for tag2 correspond to mispaired antibody chains.

[0091] In one embodiment of the ninth aspect, the at least one IgG antibody is one IgG antibody and the at least one Fab fragment is one Fab fragment.

[0092] In one embodiment of the ninth aspect, the at least one Fab fragments are two or more Fab fragments, e.g. 2, 3, 4, 5 or6 Fab fragments, wherein each fragment is preferably directed to a different epitope. In this embodiment, it is understood that in step a) each light chain of each additionally present Fab fragment is fused to a further tag, i.e. the light chain of a third Fab fragment to tag3, the light chain of a fourth Fab fragment to tag4, the light chain of a fifth Fab fragment to tag5, and so on; and that the staining in step d) is carried out with (a) detection agent(s) specific for each further tag; and that a discrimination between all tags is possible when separated according to the molecular weight.

[0093] In one embodiment of the ninth aspect, mispaired heavy and light chains are identified via their molecular weight and stain, and the ratio of the different mispaired heavy and light chain species to each other is determined by comparing the stain intensities of the respective molecular weight bands.

[0094] In one embodiment of the ninth aspect, the paired heavy and light chains are provided in step a) by contacting the chains of the IgG antibody (wherein these two chains are preferably provided in a non-paired manner) with the chains of the Fab fragment under conditions that allow for the pairing of these chains and the formation of disulfide bonds, Thus, the chains, i.e., the proteins, may be contacted with each other under the afore-mentioned conditions to arrive at paired heavy and light chains.

[0095] In one embodiment of the ninth aspect, the paired heavy and light chains are provided in step a) by expressing at least one nucleic acid encoding at least the chains of step a) in a cell (optionally resulting in the pairing of the chains and the formation of disulfide bonds) to obtain paired heavy and light chains. In this embodiment, it is understood that the at least one nucleic acid encodes the chains of all Fab fragments concerned, e.g. the chains of 3, 4, 5 or 6 Fab fragments, and the at least one IgG antibody, respectively. In this embodiment, the paired heavy and light chains may be comprised in a cell culture supernatant. The paired heavy and light chains may be purified from the cell culture supernatant, optionally to remove further components comprised in the cell culture supernatant andfurther optionally wherein the paired chains are comprised in a solution after the purification. It is understood that such a purification, if carried out, does not impact the pairing of the chains, i.e. the paired heavy and light chains remain paired heavy and light chains during the purification and corresponding conditions are chosen. Further, the heavy and light chains may be concentrated but still be comprised in cell culture supernatant or the paired heavy and light chains may be concentrated after the purification, optionally wherein the paired heavy and light chains are comprised in a solution after the concentration. It is understood that such a concentration, if carried out, does not impact the pairing of the chains, i.e. the paired heavy and light chains remain paired heavy and light chains during the concentration and corresponding conditions are chosen. In this embodiment, the at least one nucleic acid is expressed by transfecting the cell with the afore-mentioned at least one nucleic acid, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the at least one nucleic acid is expressed transiently and / or stably. In other words, a transient expression of the encoded heavy and light chains in the cells or a stable expression of the encoded heavy and light chains in the cells can be used when it comes to the expression. A stable expression may in particular be achieved by integrating corresponding expression cassettes into the genome of the cell. Furthermore, inducible expression systems may be used to be able to switch the expression on and off.

[0096] In one embodiment of the ninth aspect, the paired heavy and light chains are comprised in a solution.

[0097] In one embodiment of the ninth aspect, tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is at least about 3 kDa, preferably at least about 5 kDa, more preferably at least about 10 kDa and most preferably at least about 15 kDa. The difference between MW1 and MW2 may e.g. be about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa or about 20 kDa. Preferably, the difference between MW1 and MW2 is between about 15 kDa to about 20 kDa.

[0098] In one embodiment of the ninth aspect, tag1 and / or tag2 is / are fused to the N-terminus of the chain.

[0099] In one embodiment of the ninth aspect, the denaturing in step b) is carried out using a detergent, preferably SDS, and / or heat.

[0100] In one embodiment of the ninth aspect, step c) is carried out by a polyacrylamide gel electrophoresis (PAGE), preferably an SDS-PAGE. Any suitable PAGE may be used, including a PAGE using a gradient (polyacrylamide) gel.

[0101] In one embodiment of the ninth aspect, step d) is carried out by blotting and incubating with the detection agents, preferably a Western Blot.

[0102] In one embodiment of the ninth aspect, steps c) to d) are carried out by an SDS-PAGE followed by a Western-Blot. It is emphasized that the SDS-PAGE and the Western Blot are carried out under nonreducing conditions. Thus, in particular dithiothreitol (DTT) is not present in any of the buffers.

[0103] In one embodiment of the ninth aspect, the detection agent specific for human Fd is an antihuman Fd antibody coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme. Accordingly, the stain is preferably a fluorescent or luminescent signal. Thus, the detection agent may be an anti-human Fd antibody coupled to an enzyme, in particular horseradish peroxidase (HRP), wherein the stain is a luminescent signal after adding the HRP- substrate.

[0104] In one embodiment of the ninth aspect, the detection agent specific for tag1 is an antibody directed to tag1 coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme. Accordingly, the stain is preferably a fluorescent or luminescent signal. It can be preferred that tag1 is coupled to a fluorophore, e.g. to Alexa Fluor 488 or Alexa Fluor 647. In principle, any suitable fluorophore may be used.

[0105] In one embodiment of the ninth aspect, the detection agent specific for tag2 is an antibody directed to tag2 coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme. Accordingly, the stain is preferably a fluorescent or luminescent signal. It can be preferred that tag2 is coupled to a fluorophore, e.g. to Alexa Fluor 488 or Alexa Fluor 647. In principle, any suitable fluorophore may be used. It is understood, as defined above in the ninth aspect, that the staining of the different detection agents can be discriminated from each other, which means e.g. that, if the antibody directed to tag1 is coupled to Alexa Fluor 488, the antibody directed to tag2 would not be coupled to Alexa Fluor 488 but rather, e.g., to Alexa Fluor 647.

[0106] In one embodiment of the ninth aspect, tag1 and tag2 are selected from the group consisting of a FLAG tag, a His-tag, a RGS-His-GpL tag, a GST tag, an MBP tag, a SUMO tag, a GFP tag, a TAP tag, a TRX tag, an HA tag, a Myc tag, a V5 tag, and a CBP tag. It is understood, as defined above in the ninth aspect, that tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa and that the the difference between MW1 and MW2 is such that a discrimination between tag1 and tag2 is possible when separated according to the molecular weight. Given that the molecular weights of suitable tags are known to the skilled person, the skilled person is in a position to choose suitable tags. As an example, the skilled person is aware of the molecular weights of the afore-mentioned tags, which are as follows (given in brackets): FLAG tag (about 1 kDa), a His-tag (about 0.8 kDa), a RGS-His-GpL tag (about 19 kDa), a GST tag (about 26 kDa), an MBP tag (about 42 kDa), a SUMO tag (about 12 kDa), a GFP tag (about 26 kDa), a TAP tag (about 25 kDa), a TRX tag (about 12 kDa), an HA tag (about 1 kDa), a Myc tag (about 1 kDa), a V5 tag (about 2 kDa), and a CBP tag (about 2 kDa).

[0107] In one embodiment of the ninth aspect, tag1 is a FLAG-tag, optionally wherein the FLAG-tag has a molecular weight of about 1 kDa.

[0108] In one embodiment of the ninth aspect, tag2 is an RGS-His-GpL tag, optionally wherein the RGS-His-GpL tag has a molecular weight of about 19 kDa.

[0109] In one embodiment of the ninth aspect, tag1 is a FLAG-tag and tag2 is an RGS-His-GpL tag such that the molecular weight difference between these two tags is about 18 kDa.BRIEF DESCRIPTION OF THE FIGURES

[0110] FIG. 1 shows modular schemes illustrating the composition of the DNA-constructs used for establishment of the WB-based method for CH 1 -CL domain mispairing assessment.

[0111] FIG. 2A shows modular schemes illustrating the individual IgG and Fab antibody chains encoded by the DNAs 1-5.

[0112] FIG. 2B shows the different possibilities for correct or incorrect assembly of the individual chains after co-expression of selected DNAs.

[0113] FIG. 3A shows Western Blot (WB)-based analysis of individual chain assemblies under nonreduced conditions after transient co-transfections of the indicated DNA mixes encoding for model Ab- 1 and model Ab-2 carrying wildtype CH1 and CL domains.

[0114] FIG. 3B shows WB-based analysis of individual chains under reduced conditions after transient co-transfections of the indicated DNA mixes encoding for model Ab-1 and model Ab-2 carrying wildtype CH1 and CL domains.

[0115] FIG. 4 shows the productivity of different mixes of DNA plasmids encoding for wildtype IgG model Ab chains in combination with DNA plasmids encoding for candidate, lead or control Fab fragment model Ab chains following transient production.

[0116] FIG. 5 shows the productivity of different mixes of DNA plasmids encoding for wildtype IgG model Ab chains in combination with DNA plasmids encoding for additional candidate, lead or control Fab fragment model Ab chains following transient production.

[0117] FIG. 6A shows WB-based analysis of co-expressions of wildtype IgG model Ab1 chains assembled with individual Fab model Ab2 chains with the sequences of indicated candidate, lead, control or wildtype CH1 and CL domains.

[0118] FIG. 6B shows WB-based analysis of co-expressions of wildtype IgG model Ab1 chains assembled with additional individual Fab model Ab2 chains with the sequences of indicated candidate, lead, control or wildtype CH1 and CL domains.

[0119] FIG. 7 shows the ELISA-based detection of solely correctly assembled IgG model Ab1 and Ab2 species by selective, model Ab-specific anti-idiotype antibodies.

[0120] FIG. 8A shows an ELISA-based standard curve generated using mixes of purified model Ab1 and model Ab2 with increasing ratios of mispaired CH1-CL species spiked in.

[0121] FIG. 8B shows the positive correlation of apparent mispairing determined through measurement and actual mispairing in a scatter blot.

[0122] FIG. 9 shows the ELISA-based data from three independent experiments as well as the mean values regarding the proportions of correct chain pairing in different mixes of wildtype IgG model Ab chains in combination with candidate, lead or control IgG model Ab chains.

[0123] FIG. 10 shows data from LC-MS mispairing analysis of Lead1-3 with controls, mispairing control (MP), wildtype (WT) and Charge Variants (CV).

[0124] FIG. 11 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Leadl mutations on targetexpressing cells.

[0125] FIG. 12 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Lead2 mutations on targetexpressing cells.

[0126] FIG. 13 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Lead3 mutations on targetexpressing cells.

[0127] FIG. 14 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Lead4 mutations on targetexpressing cells.

[0128] FIG. 15 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Lead5 mutations on targetexpressing cells.

[0129] FIG. 16 shows blotted data from flow cytometric binding assay of model Ab1 and model Ab3 variants comparing antigen-specific binding of the wildtype with the Lead6 mutations on targetexpressing cells.

[0130] FIG. 17 shows the productivity of Lead7-16, Lead4, Charge Variants (CV), and DuetMab (DM) compared to the wildtype in small-scale transient transfection batches of Expi293F HEK cells.

[0131] FIG. 18 shows WB-based analysis of co-expressions of wildtype Fab model Ab2 chains assembled with individual IgG model Ab1 chains with the sequences of a first set of indicated candidate, lead, control (CV: Charge Variants; X: Domain Swap) or wildtype (WT) CH1 and CL domains.

[0132] FIG. 19 shows WB-based analysis of co-expressions of wildtype Fab model Ab2 chains assembled with individual IgG model Ab1 chains with the sequences of a second set of indicated candidate, lead, control (CV: Charge Variants; DM: DuetMab) orwildtype (WT) CH1 and CL domains.

[0133] FIG. 20 shows the ELISA-based mean values regarding the proportions of correct chain pairing in different mixes of wildtype IgG model Ab chains in combination with a first set of indicated candidate, lead or control IgG model Ab chains (WT: wildtype; MP1 / MP2: mispairing controls; X: Domain Swap; CV: Charge Variants; DM: DuetMab).

[0134] FIG. 21 shows the ELISA-based mean values regarding the proportions of correct chain pairing in different mixes of wildtype IgG model Ab chains in combination with a second set of indicated candidate, lead or control IgG model Ab chains (WT: wildtype; MP1 / MP2: mispairing controls; X: Domain Swap; CV: Charge Variants; DM: DuetMab).

[0135] FIG. 22 shows the results of the LC-MS analysis regarding the proportions of correct chain pairing in different mixes of wildtype IgG model Ab chains in combination with a first set of indicated lead or control IgG model Ab chains (WT: wildtype; MP1 / MP2: mispairing controls).

[0136] FIG. 23 shows the results of the LC-MS analysis regarding the proportions of correct chain pairing in different mixes of wildtype Fab model Ab chains in combination with a second set of indicated lead or control Fab model Ab chains (DM: DuetMab).

[0137] FIG. 24 shows the results of a flow cytometric binding assay regarding the retained antigenspecific binding of model Ab1 variants containing the Lead7-11 mutations in the CH1 and CL domains compared to the wildtype model Ab1 antibody.

[0138] FIG. 25 shows the results of a flow cytometric binding assay regarding the retained antigenspecific binding of model Ab1 variants containing the Lead12-16 mutations in the CH1 and CL domains compared to the wildtype model Ab1 antibody.DETAILED DESCRIPTION OF THE INVENTION

[0139] The present invention is inter alia directed to antibodies or fragments thereof as well as fusion proteins comprising the same, and nucleic acids encoding the afore-mentioned antibodies or fragments thereof or fusion proteins, wherein a CH1 domain and a CL domain comprise the specific amino acid substitutions as disclosed herein. The aim of these amino acid substitutions is to ensure a pairing of the resulting CH1 domain with the resulting CL domain such that mispairing with further CH1 and CL domains (without these amino acid substitutions, e.g. wild-type CH1 and CL domains) is prevented. The concept of “preferential pairing” between CH1 and CL domains comprising the substitutions as disclosed herein is particularly relevant for multispecific (such as e.g. bispecific) antibodies or fragments thereof or fusion proteins, and for mixtures of at least two different antibodies or fragments thereof or fusion proteins, and for nucleic acids encoding the multispecific antibodies or fragments thereof or fusion proteins or encoding mixtures of at least two different antibodies or fragments thereof or fusion proteins.

[0140] Thus, the following substitutions in a CH1 domain and a CL domain or at least one nucleic acid encoding a CH1 domain and a CL domain comprising the following substitutions are relevant for the present invention:(i) amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU in the CH1 domain, and (a)(ii) amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (b)(i) amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU in the CH1 domain, and (b)(ii) amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU in the CL domain;(i) amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU in theCH1 domain, and (ii) amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU in the CL domain;(i) amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU in the CL domain;(i) amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU in the CL domain;(i) amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU in the CL domain;(i) amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU in the CL domain;(i) amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU in the CL domain;(i) amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU in the CH1 domain, and (a)(ii) amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU in the CL domain;(i) amino acid substitutions (i’) T 137W, T 173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or(i”) G141W and K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’”) A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU in the CL domain; and(i) amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU in the CH1 domain, and (ii) amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU in the CL domain.Definitions

[0141] As used herein, the singular form of “a” or “an” also includes the corresponding plural unless the context clearly dictates otherwise.

[0142] The term “about” in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10% and preferably ±5%.

[0143] It needs to be understood that the term “comprising” is not limiting. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

[0144] The suffix “n” in the context of peptides and antibody formats as used herein refers to the number of moieties, entities, domains etc. that are present in the antibody or fragment thereof or fusion protein wherein n refers to naturals such as 1 , 2 ,3, 4, etc.

[0145] The term “nucleic acid” as used herein is defined as a chain of nucleotides and interchangeably used with the term "polynucleotide". A skilled person has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e . , the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means. The term "polynucleotide" as used herein is to be interpreted broadly, and includes DNA and RNA, including modified DNA and RNA.

[0146] The nucleic acid as defined herein is preferably “isolated", which means that the nucleic acid is altered or removed from the natural state. For example, a nucleic acid (or a peptide) naturally present in a living animal is not "isolated", but the same nucleic acid (or peptide) partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid (orpeptide) can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell or it can be present in a host cell that has been genetically modified to express the isolated nucleic acid.

[0147] The term “encodes" or “encoding” as used herein refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, mRNA, or miRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. For the present invention, the synthesis of proteins, more particularly antibodies or fragments thereof or fusion proteins comprising the same (all of which have a defined sequence of amino acids) is more relevant. On a general level, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0148] The term “DNA” as used herein is the usual abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine- monophosphate and deoxy-cytidine-monophosphate monomers or analogs thereof which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate-backbone, is called the DNA-sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A / T-base-pairing and G / C-base- pairing.

[0149] The term “RNA” as used herein relates to a nucleic acid molecule which includes ribonucleotide monomers. In preferred embodiments, the RNA contains all or a majority of ribonucleotide monomers. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, without limitation, 5- methylcytidine, pseudouridine and / or 1-methyl-pseudouridine. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine. In some embodiments,the RNA according to the present disclosure comprises a 5'-cap. In one embodiment, the RNA of the present disclosure does not have uncapped 5'-triphosphates. In one embodiment, the RNA may be modified by a 5'- cap analog. The term "5'-cap" refers to a structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via a 5' to 5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. Providing an RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription, in which the 5'-cap is co- transcriptionally expressed into the RNA strand, or may be attached to RNA post-transcriptionally using capping enzymes.

[0150] The term “mRNA” as used herein relates to an RNA transcript which encodes a peptide or protein. As established in the field, mRNA generally contains a cap structure, a 5' untranslated region (5'-UTR), a peptide coding region, a 3' untranslated region (3'-UTR) and a polyA tail. In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides. These methods are known by the skilled person in the art.

[0151] The term “chemically modified” with respect to a nucleic acid or DNA or RNA, respectively, as used herein refers to chemically modified nucleotides, such as e.g. the above-mentioned ribonucleotides in the RNA definition. The term also includes any chemical modifications in the phosphate-groups linking the nucleotides.

[0152] The term “antibody” as used herein refers to a glycoprotein belonging to the immunoglobulin superfamily. The term is intended herein to refer to a full-length antibody, i.e. an immunoglobulin molecule that binds to a target molecule and contains four peptide chains: two heavy chains and two light chains which are connected to each other through disulfide bonds. An antibody may recognise an antigen via the variable region of the antigen-binding fragment (Fab). The fragment crystallizable region (Fc region) is the tail region of an antibody that may allow antibodies to activate the immune system and may affect the plasma half-life. The hinge region is a stretch of the heavy chains linking the Fab and Fc regions. The heavy chain and light chain typically each comprise a variable region and one or more constant domains. For example, in IgG antibodies, a heavy chain comprises a variable region (VH) and three constant domains (CH1 , CH2, and CH3) and a light chain comprises a variable region (VL) and one constant domain (CL). Examples of antibodies include a mono- or a multispecific antibody, which in turn can be monoclonal or polyclonal, human, humanized, chimeric antibody. Antibodies can come in different varieties known as isotypes or classes. In humans there are five antibody classes known as IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses such as lgG1 , lgG2, lgG3, and lgG4. The prefix "Ig" stands for immunoglobulin, while the suffix denotes the type of heavy chain the antibody contains: the heavy chain types a (alpha), y (gamma), 5 (delta), E (epsilon), p (mu) give rise to IgA, IgG, IgD, IgE, IgM, respectively. The distinctive features of each class are determined by the part of the heavy chain within the hinge and Fc region. Preferably, the antibodies of the present disclosure are IgG antibodies and even more preferably IgG 1 antibodies.

[0153] The term “symmetric antibody” as used herein refers to the classical setup of e.g. a full-length antibody that comprises two identical antigen-binding regions (or two identical arms) such that the antibody is monospecific.

[0154] The term “asymmetric antibody” as used herein refers to an antibody with at least two different antigen-binding regions (or at least two different arms) such that the antibody is multispecific.

[0155] The term “human antibody” as used herein refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

[0156] The term “humanized antibody” as used herein refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0157] The term “chimeric antibody” as used herein refers to an antibody in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0158] The term “epitope” as used herein refers to the part of an antigen that binds to an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0159] The term “anti-idiotype antibody” as used herein refers to an antibody that binds to the variable region of another antibody (wherein the “another antibody” is the idiotype). As used herein, the term refers to anti-idiotype antibodies that bind to the variable region of an antibody in a manner that the correct assembly of the heavy and the light of this antibody (i.e. of the idiotype) are required for binding of the anti-idiotype antibody.

[0160] The term “detection agent specific for human Fd” or more specifically “anti-human Fd antibody” as used herein refers to an agent or antibody, respectively, which specifically binds to the Fd region of human IgG, which corresponds to the first about 220 amino acids from the N-terminus of the heavy chain, contained within the Fab’ region of a human IgG.

[0161] The term “CH1” as used herein means constant heavy chain 1 , preferably human constant heavy chain 1 . The CH1 domain is comprised in the heavy chain of an antibody.

[0162] The term “CL” as used herein means constant light chain, preferably human constant light chain. The CL domain is comprised in the light chain of an antibody. The CL domain may be of kappa isotype or it may be of lambda isotype, preferably kappa isotype.

[0163] The term “a CH 1 domain matches a CL domain” as used herein means that the corresponding CH1 and CL domains are present in conformations, which are suitable for assembly with each otheror even facilitate such an assembly. In other words, the respective CH1 and the CL domains fit with each other and assemble to form at least a fragment comprising these two domains. Such “matching” or “fitting” CH1 and CL domains may e.g. be the CH1 and CL domains of the present invention but also e.g. a wild-type CH1 domain and a wild-type CL domain.

[0164] The term “fragment” as used herein refers to a fragment of an antibody, which fragment may be involved in binding with the target molecule. Examples of such antibody fragments include an antigen-binding fragment (Fab), a Fab', a Fab'-SH, a fragment antibody F(ab’)2, a variable region (Fv), a single chain antibody (scFv), a single-domain antibody (sdAb), and a camelid antibody (VHH). The term “Antigen-binding fragment” or “Fab” refers to a region of an antibody that binds to antigens and is composed of one constant and one variable region of each of the heavy and the light chain. The term “fragment antibody” or “F(ab’)2” refers to a region of an antibody that remains following digestion of the Fc region while leaving intact some of the hinge region. The term “Fab”’ refers to a fragment formed by the reduction of a F(ab')2 fragment. However, a fragment in accordance with the present invention must not be involved in binding with the target molecule and can e.g. be a CH1-CL fragment. Such a CH1-CL fragment may in particular be a fused CH1-CL-domain.

[0165] The term “Kabat” as used herein refers to an immunoglobulin alignment and numbering system pioneered by Elvin a. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. or Kabat et al., 1992, Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719).

[0166] The term “EU” as used herein refers to an immunoglobulin alignment and numbering system pioneered by Edelman et al. (Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(1):78-85).

[0167] The amino acids as referred to herein are typically indicated herein by commonly known one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The term “amino acid substitution” as used herein means that a specific amino acid at a specific position is replaced by another amino acid. If the amino acid substitution is e.g. “S136C”, this means that the S at position 136 (here according to e.g. the Kabat numbering) is replaced by and thus substituted by a C. A way of easily introducing such substitutions is the amendment of the corresponding coding sequence in the encoding nucleic acid such that the expressed peptide already contains the substituted amino acid. This in particular applies to the at least one nucleic acid encoding at least a CH1 domain and a CL domain comprising such substitutions.

[0168] The terms "amino acid" and "amino acid residue" are used herein interchangeably. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids may be classified based on structure and chemical characteristics. Thus, classes of amino acids may be reflected in Table A below:Table A: Classification based on structure and general chemical characterization of R group

[0169] When it comes to the amino acid substitutions as disclosed and claimed herein, a replacement and thus substitution of a specific amino acid to a different amino acid at the respective position (e.g., C233V, meaning that the C at position 233 is replaced by a V) is meant to encompass not only the substitution to the specific amino acid as indicated (in the above example the V) but also to another amino acid of the corresponding class according to Table A (in the above example, the class of “aliphatic uncharged Residues” comprising V further comprises G, A, L and I such that the following substitutions are meant to be encompassed in addition to the C233V substitution: C233G, C233A, C233L and C233I). Another example is the replacement resulting in a D or an E at a specific position: D and E are both in the class of “Acidic Residues” such that both are meant to be encompassed if the amino acid resulting from the substitution is a D or an E. The afore-mentioned possibilities of varying within a given class of amino acids is typically referred to as a “conservative amino acid exchange”. Importantly, this does not apply if the amino acid after the substitution is a C at the corresponding position (e.g., S134C in CH1 according to Kabat I S136C according to EU in the CH1 domain; or S114C according to Kabat and EU in the CL domain). In the latter scenario, M and P of the class “Nonpolar Uncharged Residues” are not meant to be encompassed by indicating a C as the amino acid resulting from the substitution.

[0170] The term “peptide” as used herein refers to a compound comprised of amino acid residues covalently linked by peptide bonds and is used interchangeably with the terms “protein” and “polypeptide”. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. In some embodiments, the maximum number of amino acids of the peptide or protein is about 700 to 1000 amino acids. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0171] By "wild-type" or "WT" or "native" herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild-type amino acid sequence, peptide or protein has an aminoacid sequence that has not been intentionally modified. This definition in particular applies for the “wildtype CH1 domain” and the “wild-type CL domain”.

[0172] The term “fusion protein” as used herein means that an antibody comprising the CH1 and CL domains of the present invention or a fragment thereof comprising or consisting of the CH1 and CL domains of the present invention is fused to a peptide, typically via a linker peptide. The peptide may be fused N-terminally or C-terminally or two peptides may be fused, one N-terminally and one C- terminally (which applies in the case of antibodies twice because there are two heavy chains and two light chains). If two or more peptides are present in fusion proteins comprising an antibody or fragment thereof, the peptides may be the same peptide, e.g. a cytokine. Alternatively, the peptides may be different, e.g. a cytokine and a receptor. Moreover, the antibody or fragment thereof of the fusion protein may comprise at least two chains with an N-terminus and a C-terminus per chain. The peptide may by terminally fused either to the N-terminus or the C-terminus or to both termini of the at least two chains. For example, in an antibody with an IgG format with four chains, there are 8 termini (2 N- termini of the light chains, 2 N-termini of the heavy chains, 2 C-termini of the light chains and 2 C- termini of the heavy chains) and a peptide can be fused to one, two, three, four, five, six, seven or all eight termini. In one embodiment the peptide is only fused to the C-termini of the antibody or fragment thereof.

[0173] The peptide that may be fused can e.g. be a cytokine, a receptor or a ligand, wherein these terms are used in the meaning as commonly known to the skilled person. Thus, a cytokine is typically a small protein involved in cell signaling, whereas a receptor is typically a cell membrane-spanning or cell membrane-bound protein, which is also involved in cell signaling, and wherein a ligand binds to a receptor, typically to evoke or induce a certain signal or signaling pathway. For the present invention, a peptide can in particular also be an antibody fragment as discussed above, which provides a toolbox of designing multispecific antibodies by fusing e.g. antigen-binding antibody fragments to an antibody or a fragment thereof.

[0174] The term "pharmaceutically acceptable" as used herein refers to molecular entities and other ingredients (mainly of compositions) that are physiologically tolerable and do not produce undesired reactions when administered to a mammal (e.g., human). The term "pharmaceutically acceptable" can also mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. A "pharmaceutically acceptable carrier and / or excipient" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject.

[0175] The term "vector" as used herein refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. A "lentivirus" as usedherein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells. They can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0176] The term "treatment" or "treating" as used herein relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of a therapeutically effective composition to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0177] The term “ELISA” as used herein refers to the ELISA-method as generally known to the skilled person. Thus, it refers to an enzyme-linked immunosorbent assay, which is a solid-phase type of enzyme immunoassay to detect the presence of a ligand (commonly a protein, in the present disclosure primarily an antibody) in a liquid sample using antibodies directed against the ligand to be measured. In the simplest form of an ELISA, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate is added. If there was binding, the subsequent reaction produces a detectable signal, most commonly a color change. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen (the “capture reagent”), in a "sandwich" ELISA, which is primarily relevant for the present disclosure). After the antigen is immobilized, the detection antibody (the “detection agent”) is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or a dye (in particular a fluorescent dye) or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal or the signal of the dye (in particular the fluorescence of a fluorescent dye) is detected, which indicates the quantity of antigen in the sample. The term as used herein includes a manual ELISA, wherein (semi-)automated ELISA systems are preferred, such as, e.g., GyroLab.

[0178] The term “PAGE” as used herein refers to a common polyacrylamide gel electrophoresis as well known to the skilled person. An “SDS-PAGE” is a discontinuous electrophoretic system commonly used as a method to separate proteins with molecular masses between about 5 and about 250 kDa. The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) andpolyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. Gradient gels may be used in a PAGE.

[0179] The term “Western Blot” as used herein refers to a common method relying on immunodetection. A Western Blot relies on proteins separated by size (typically achieved by an SDS- PAGE, transfer of the proteins to a solid support (in particular a membrane), and staining the proteins using either a primary and secondary antibody to visualize or a primary antibody coupled to a dye (in particular a fluorophore) or an enzyme. On a general level, detection can be by various methods such as staining, fluorescence, luminescence and radioactivity, allowing indirect detection of the specific target protein.Particles

[0180] In some embodiments, an agent to be delivered to a subject, e.g., a nucleic acid, a polypeptide, a small molecule, and the like, is encapsulated in a particle. In some embodiments, the at least one nucleic acid of the present disclosure is formulated in (e.g., encapsulated in) a particle, as further described herein. In some embodiments, a particle is a nucleic acid particle wherein the nucleic acid particle comprises a nucleic acid (e.g., DNA and / or RNA), and a cationic lipid, a cationically ionizable lipid, or a cationic polymer.

[0181] A “nucleic acid particle,” as used herein, refers to a particle that encompasses or contains a nucleic acid, and, is part of a composition (e.g., a pharmaceutical composition) comprising multiple nucleic acid particles, that is useful for (i) enhancing nucleic acid stability, e.g., during storage, (ii) improving biodistribution of the nucleic acid or delivering a nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like), and / or (iii) facilitating cell uptake of the nucleic acid. As described herein, a nucleic acid particle may be formed from i) at least one cationic or cationically ionizable lipid or lipid-like material; ii) at least one cationic polymer such as polyethyleneimine, protamine, or a mixture thereof (i.e. , a mixture of i) and ii)), and iii) a nucleic acid. Nucleic acid particles described herein include lipid nanoparticles (LNP), lipoplexes (LPX), liposomes, and polyplexes (PLX).

[0182] Electrostatic interactions between positively charged molecules such as cationic polymers and cationic lipids and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. The characteristics of a particle (e.g., nanoparticle) are determined, at least in part, from the components used to form the particle and the process used to prepare the particle. A description of the different types of particles and their structures is provided in ACS A / ano 2021 , 15, 11 , 16982-17015.

[0183] In some embodiments, a nucleic acid particle described herein is a nanoparticle. As used in the present disclosure, “nanoparticle” refers to a particle having an average diameter suitable for parenteral administration and is less than 1000 nm in diameter. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 120 nm, about 50 nm to about 100 nm, or about 60 nm to about 90 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 40 nm to about 120 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamiclaser light scattering (DLS) with data analysis using an appropriate algorithm (e.g., the so-called cumulant algorithm for monodisperse samples), which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0184] A composition comprising nucleic acid particles can be characterized by its polydispersity index, that is, the relative uniformity of particles within a given composition. For example, compositions described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less of said nanoparticles. In some embodiments, a composition comprising nucleic acid particles, as described herein, may exhibit a PDI less than about 0.3. By way of example, a composition comprising nucleic acid particles described herein can exhibit a PDI in a range of about 0.1 to about 0.3, or about 0.2 to about 0.3. The polydispersity index of a given composition can be calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles.

[0185] Nucleic acid particles described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic lipid or polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in permanently cationic form (e.g., N+), or one that is ionizable to become cationic (e.g., under certain pH conditions). Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1 , e.g., an N / P ratio of about 4 is intended to mean about 4:1. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 4. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is less than 24, less than 18, or less than 12. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is from about 2 to about 24, about 4 to about 18, about 4 to about 12, or about 4 to about 8. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., an RNA LNP) described herein is about 6.

[0186] Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. As used herein, an “ionizable” lipid, e.g., a “cationically ionizable” lipid or “ionizable” polymer, e.g., a “cationically ionizable” polymer is a lipid or polymer that may be, in some embodiments, neutral at physiological pH, but is capable of becoming cationic (i.e., becoming positively charged) at acidic pH.

[0187] The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and / or at least one cationic polymer which associate with the nucleic acid to form nucleic acid particles (e.g., RNA nanoparticles) and compositionscomprising such particles. The nucleic acid particles (e.g., RNA nanoparticles) may comprise nucleic acid which is complexed by different non-covalent interactions (e.g. electrostatic, hydrogen bonding, pi-stacking, van der Waals, etc.) to the particle. In some embodiments, the particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e., they are not able to virally infect cells.

[0188] In a nucleic acid particle (e.g., RNA nanoparticle) composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. In some embodiments, a composition comprises more than one individual nucleic acid particle (e.g., RNA nanoparticle) formulation. Respective pharmaceutical compositions are referred to as “mixed particulate formulations.” Such mixed particulate formulations may be obtainable by forming, separately, individual nucleic acid particle formulations, and mixing these to produce a formulation comprising a mixed population of nucleic acid-containing particles. Alternatively, different nucleic acid species may be formulated together as a “combined particulate formulation.” Such formulations may be obtainable by mixing a combined formulation of different nucleic acid species with a particle-forming agent, to produce particles that comprise more than one nucleic acid species.Lipid Nanoparticles (LNPs)

[0189] In some embodiments, a particle described herein is a lipid nanoparticle (LNP). LNPs have emerged as particularly useful vehicles for delivery of nucleic acids, for example as described in Theranostics, 2022 Oct 24;12(17):7509-7531. It is understood that a LNP is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex. LNPs, as described herein, typically do not comprise a bilayer (uni-lamellar), or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous compartment. Moreover, LNPs, as described herein, typically do not comprise a central aqueous core or compartment. LNPs as described herein typically comprise nucleic acids (e.g., DNA or RNA such as mRNA) and lipids forming a disordered, non-lamellar phase. LNPs as described herein may be considered as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. See, e.g., ACS Nano 2021 , 15, 11 , 16982- 17015; Aldosari, et al., Pharmaceutics, 2021 , 13, 206.

[0190] LNPs described herein generally comprise four categories of lipids in addition to a nucleic acid agent: a cationically ionizable lipid (typically a cationically ionizable lipid), a polymer-conjugated lipid, a helper lipid, and a steroid. A person of skill in the art will understand that various combinations of these four categories of lipids can be used to prepare lipid nanoparticles for use in delivering nucleic acid agents.Cationic or cationically ionizable lipids

[0191] As described generally herein, a nucleic acid particle comprises a nucleic acid and a cationic or a cationically ionizable lipid. In some embodiments, a cationic or cationically ionizable lipid useful for incorporation into a nucleic acid particle are those lipids having a polar head group and an aliphatictail. In some embodiments, a cationic lipid is one where the polar head group has a permanently positive charge (for example, comprising a quaternary ammonium group). In some embodiments, a cationically ionizable lipid is a lipid wherein, at a given pH and in the context of an LNP, the lipid becomes positively charged, such as at below physiological pH (e.g., below pH about 7.4) or neutral pH (e.g., a pH around 7 to 7.5), or in some embodiments, at a pH of less than 7 (e.g., less than 6). In some embodiments, a cationically ionizable lipid is one comprising polar head group that comprises one or more a tertiary amine groups (or secondary or primary amine group) that can become positively charged. LNPs typically comprise cationically ionizable lipids.

[0192] Suitable cationic or cationically ionizable lipids are readily identified by those of skill in the art. In some embodiments, a cationic lipid or cationically ionizable lipid is one provided in W02012 / 016184, which is incorporated herein by reference in its entirety.

[0193] In some embodiments, a cationically ionizable lipid is a lipid described in WO2017 / 075531 or WO2018 / 081480, each of which is incorporated by reference herein in its entirety.

[0194] In some embodiments, a cationic lipid is one described in WO2017 / 049245, which is incorporated by reference in its entirety.

[0195] In some embodiments, a cationically ionizable lipid is a lipid described in WO2015 / 095340, which is incorporated by reference herein in its entirety.

[0196] In some embodiments, a cationic lipid is one described in WO2018 / 087753, which is incorporated herein by reference in its entirety.Helper lipids

[0197] As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid (also referred to as a neutral lipid). In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is a phospholipid.

[0198] Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO2021 / 026358, WO 2017 / 075531 , and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference.Polymer conjugated lipids

[0199] As described herein, LNPs of the present disclosure comprise a polymer-conjugated lipid. In some embodiments, a polymer conjugated lipid is a lipid conjugated to polyethylene glycol (a “PEG- lipid”).

[0200] In some embodiments, a PEG-lipid is provided in WO2021 / 026358, WO 2017 / 075531 , or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[0201] In some embodiments, an LNP comprises a polysarcosine-conjugated or a pAEEA / pMAEEA- conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).Steroids

[0202] As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol.Manufacturing

[0203] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Publication Nos. 2016 / 0009637, 2015 / 0273068, 2014 / 0200257, 2013 / 0338210, 2013 / 0245107, 2013 / 0123338, 2013 / 0017223,2012 / 0183581 , 2012 / 0027803, 2011 / 0311583, 2011 / 0216622, 2011 / 0117125, 2007 / 0042031 ,2006 / 0083780, 2005 / 017054, 2004 / 0142025, 2007 / 0042031 , 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741 , WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531 , WO 2015 / 199952, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, WO 2022 / 016089, WO 2022 / 081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.Liposomes

[0204] In some embodiments, a nucleic acid particle is a liposome, wherein the liposome comprises a cationic lipid and a nucleic acid. Liposomes are lipid-based particles that comprise a bilayer (unilamellar) or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous core that encapsulates the agent for delivery (e.g., a nucleic acid such as RNA). Different types of liposomes are described, including e.g., small and large unilamellar vesicles, multilamellar vesicles, multivesicular liposomes. Many suitable methods are known for manufacturing liposomes (see e.g., Shah S, et al., Adv Drug Deliv Rev. 2020;154-155:102- 122), including e.g., solvent evaporation or lipid film hydration, solvent dispersion or reverse phase evaporation, optionally followed by processes to manipulate the size of the liposomes, such as e.g., sonication, homogenization and extrusion. Examples of liposomes that may be suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO2012 / 006378, WO2013 / 006825, WO2019 / 077053 and WO2022 / 069632, each of which is incorporated herein by reference in its entirety.Lipoplexes (LPX)

[0205] In some embodiments, a nucleic acid particle is a lipoplex, wherein the lipoplex comprises a cationic lipid and a nucleic acid. Lipoplex particles (LPX) may be prepared by mixing liposomes with nucleic acid (e.g., RNA, where lipoplex particles comprising RNA are referred to as “RNA lipoplex particles”). RNA LPX particles typically form spontaneously from electrostatic interactions between positively charged liposomes and negatively charged RNA, and typically have a multilamellar structure. LPX (e.g., RNA LPX) typically comprise one or more cationic lipids and optionally one or more additional lipids. Examples of lipoplexes that are suitable for nucleic acid (e.g., RNA) delivery, as well as methods of manufacture, are described in PCT App. Pub. No. WO2019 / 077053 and WO2022 / 069632, each of which is incorporated herein by reference in its entirety.Polymer-based particles (Polyplexes) and other delivery systems

[0206] In some embodiments, a nucleic acid particle described herein is a polymer-based particle (i.e., a polyplex, PLX). In some embodiments, a nucleic acid particle is a polyplex particle, and comprises a cationic polymer and a nucleic acid. Examples of polyplex particles that are suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO 2021 / 001417, which is incorporated herein by reference in its entirety.Numbered embodiments

[0207] The present invention also relates to the following embodiments:1 . An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU; preferably (a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU, and (a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU, and (b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU.2. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU.3. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143Wand H168D in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118A, N137Qand D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU.4. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU.5. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU.6. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU.7. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU.8. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU; preferably (a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174Cand C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU, and (a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU.9. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH 1 ) domain comprising the amino acid substitutions (i’) T 137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or (i”) G141W and K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’”) A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU.10. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH 1 ) domain comprising the amino acid substitutions H 172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU.11 . The antibody or fragment thereof according to any one of embodiments 1 to 10, wherein the antibody or fragment thereof is an IgG antibody or IgG antibody fragment thereof.12. The antibody or fragment thereof according to any one of embodiments 1 to 11 , wherein the antibody or fragment thereof is an IgG 1 antibody or IgG 1 antibody fragment thereof.13. The antibody or fragment thereof according to any one of embodiments 1 to 12, wherein the CL domain is of kappa isotype.14. The antibody or fragment thereof according to any one of embodiments 1 to 13, wherein the antibody or fragment thereof is an antibody selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.15. The antibody or fragment thereof according to any one of embodiments 1 to 13, wherein the antibody or fragment thereof is a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.A fusion protein comprising the antibody or fragment thereof according to any one of embodiments 1 to 15. The fusion protein according to embodiment 16, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof. The fusion protein according to embodiment 16 or 17, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody- peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1- CL), CH1-CL-peptidei-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2) . The fusion protein according to embodiment 17 or 18, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH. The fusion protein according to any one of embodiments 16 to 19, wherein the fusion protein is selected from the group consisting of symmetric IgG antibody-cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab-cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2-VHH2, cytokine- CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1-CL-VHH, scFv- CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2-CH1-CL- SCFV2, VHH2-CHI-CL-VHH2, scFv2-CH1-CL-VHH2and VHH2-CH1-CL-scFv2. The antibody or fragment thereof according to any one of embodiments 1 to 13, wherein the antibody or fragment thereof comprises at least two different CH1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are (i) and (ii), and wherein each additional CH1 domain comprises a CH1 domain different from (i) and each additional CL domain comprises a CL domain different from (ii), wherein the antibody or fragment thereof preferably comprises two different CH1 domains and two different CL domains. The antibody or fragment thereof according to embodiment 21 , wherein each additional CH1 domain matches an additional CL domain. The antibody or fragment thereof according to embodiment 21 or 22, wherein an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain. The antibody or fragment thereof according to any one of embodiments 21 to 23, wherein the antibody or fragment thereof is selected from the group consisting of an asymmetric IgGantibody and a F(ab’)2 fragment.25. A fusion protein comprising the antibody or fragment thereof according to any one of embodiments 21 to 24.26. The fusion protein according to embodiment 25, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof.27. The fusion protein according to embodiment 25 or 26, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) asymmetric IgG antibody-peptide2 and F(ab’)2-peptide2.28. The fusion protein according to embodiment 26 or 27, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.29. The fusion protein according to any one of embodiments 25 to 28, wherein the fusion protein is selected from the group consisting of asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, and F(ab’)2- VHH2.30. A mixture of at least two antibodies and / or fragments thereof and / or fusion proteins comprising an antibody or fragment thereof, wherein a first antibody or fragment thereof is the antibody or fragment thereof according to any one of embodiments 1 to 15 or a first fusion protein is the fusion protein according to any one of embodiments 16 to 20 [wherein each CH1 domain and each CL domain of the corresponding antibody or fragment thereof or fusion protein is (i) and (ii) of any one of embodiments 1 to 13]; and wherein each CH1 domain and each CL domain of each additional antibody or fragment thereof or additional fusion protein comprises a CH1 domain different from (i) of any one of embodiments 1 to 13 and a CL domain different from (ii) of any one of embodiments 1 to 13, preferably wherein the mixture comprises two antibodies and / or fragments thereof and / or fusion proteins.31. The mixture according to embodiment 30, wherein each additional antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additional antibody or fragment thereof or fusion protein.32. The mixture according to embodiment 30 or 31 , wherein an additional antibody or fragment thereof or fusion protein comprises a wild-type CH1 domain and a wild-type CL domain.33. At least one nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 15 and 21 to 24, the fusion protein according to any one of embodiments 16 to 20 and 25 to 29, or the mixture according to any one of embodiments 30 to 32.34. The at least one nucleic acid according to embodiment 33, wherein the nucleic acid is DNA or RNA, preferably mRNA.35. A composition comprising (i) the antibody or antigen-binding fragment thereof according to any of embodiments 1 to 15 and 21 to 24, (ii) the fusion protein according to any of embodiments 16 to 20 and 25 to 29, (iii) the mixture according to any of embodiments 30 to 32, or (iv) the at least one nucleic acid according to embodiments 33 and 34.36. The composition according to embodiment 35, wherein the composition is a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.37. The composition according to embodiment 35 or 36 for use in therapy.38. The composition according to any one of embodiments 35 to 37 for use in the treatment of cancer.39. The composition according to any one of embodiments 35 to 37 for use in the treatment of an infectious disease.40. The composition according to any one of embodiments 35 to 37 for use in the treatment of an autoimmune disease.41. The composition according to embodiment 35, wherein the composition is a diagnostic composition.42. Use of (i) the antibody or antigen-binding fragment thereof according to any of embodiments 1 to 15 and 21 to 24, (ii) the fusion protein according to any of embodiments 16 to 20 and 25 to 29, or (iii) the mixture according to any of embodiments 30 to 32 in diagnosis.43. A method for the preparation of an antibody or fragment thereof according to any one of embodiments 1 to 15 and 21 to 24 or of a fusion protein according to any one of embodiments 16 to 20 and 25 to 29, wherein the method comprises the steps of(i) transforming a host cell with at least one nucleic acid encoding the antibody or fragment thereof or the fusion protein;(ii) culturing the host cell under conditions allowing the synthesis of the antibody or fragment thereof or the fusion protein; and(iii) recovering the antibody or fragment thereof or the fusion protein from the culture.44. A method for the preparation of a mixture according to any of embodiments 30 to 32, wherein the method comprises the steps of(i) transforming a host cell with at least one nucleic acid encoding the mixture;(ii) culturing the host cell under conditions allowing the synthesis of the mixture; and(iii) recovering the mixture from the culture.45. Use of the following substitutions in a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain to induce a preferential binding between the respective CH1 domain and the respective CL domain:(i) the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU in the CH1 domain, and (a)(ii) the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (b)(i) the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU in the CH1 domain, and (b)(ii) the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU in the CL domain;(i) the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU in the CL domain;(i) the amino acid substitutions S130M, G141Wand H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU in the CL domain;(i) the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU in the CL domain;(i) the amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabatcorresponding to F118G, E123K and S131 D in the numbering according to EU in the CL domain;(i) the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU in the CL domain;(i) the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU in the CL domain;(i) the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU in the CH1 domain, and (a)(ii) the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU in the CL domain;(i) the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T 139W, T 169V and V185W in the numbering according to EU or (i”) G141 Wand K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’”) A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S1311 and V185W in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU in the CL domain; or(i) the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU in the CL domain.46. The use according to embodiment 45, wherein the CH1 domain and the CL domain are comprised in an antibody or a fragment thereof or a fusion protein comprising said antibody or fragment thereof.

[0208] Additionally, the present invention relates to the following numbered embodiments:At least one nucleic acid encoding at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU; preferably (1a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU, and (1 a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (1 b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU, and (1 b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU;(2)(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU, and (2)(ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU;(3)(i) a CH1 domain comprising the amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU, and (3)(ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU;(4)(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU, and (4)(ii) a CL domain comprising the amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU;(5)(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU, and (5)(ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in thenumbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU;(6)(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU, and (6)(ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU;(7)(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU, and (7)(ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU;(8)(i) a CH1 domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F 170C and C220V in the numbering according to EU, and (8)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU; preferably (8a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU, and (8a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU;(9)(i) a CH1 domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or (i”) G141 Wand K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’") A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU, and (9)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU; and(10)(i) a CH1 domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (10)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU. The at least one nucleic acid according to embodiment 1 , wherein the at least one nucleic acid encodes an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the CH1 domain and the CL domain, or a fusion protein comprising said antibody or fragment thereof.3. The at least one nucleic acid according to embodiment 1 or 2, wherein the at least one nucleic acid encodes an IgG antibody or IgG antibody fragment thereof.4. The at least one nucleic acid according to any one of embodiments 1 to 3, wherein the at least one nucleic acid encodes an IgG 1 antibody or IgG 1 antibody fragment thereof.5. The at least one nucleic acid according to any one of embodiments 1 to 4, wherein the CL domain is of kappa isotype.6. The at least one nucleic acid according to any one of embodiments 2 to 5, wherein the at least one nucleic acid encodes an antibody selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.7. The at least one nucleic acid according to any one of embodiments 2 to 5, wherein the at least one nucleic acid encodes a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.8. The at least one nucleic acid according to any one of embodiments 2 to 5, wherein the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C- terminus of the antibody or fragment thereof.9. The at least one nucleic acid according to any one of embodiments 2 to 5 or 8, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody-peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptidei-2 (preferably CH 1-CL-peptide2) and peptide-1-2- CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2) .10. The at least one nucleic acid according to embodiment 8 or 9, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.11 . The at least one nucleic acid according to any one of embodiments 2 to 5 or 8 to 10, wherein the fusion protein is selected from the group consisting of symmetric IgG antibody-cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody- cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab-cytokine, Fab- scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2-VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1-CL- VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2- CH 1 -CL-SCFV2, VH H2-CH 1 -CL-VHH2, scFv2-CH 1 -CL-VH H2and VH H2-CH 1 -CL-scFv2.12. The at least one nucleic acid according to any one of embodiments 1-5, wherein the at least one nucleic acid encodes an antibody or fragment thereof, or a fusion protein comprising said antibody or fragment thereof, wherein the antibody or fragment thereof comprises at least two different CH1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are selected from the group consisting of (1)(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each additional CH1 domain comprises a CH1 domain different from (1)(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and each additional CL domain comprises a CL domain different from (1 )(ii), (2)(ii), (3)(ii), (4)(ii), (5)(ii), (6)(ii), (7)(ii), (8)(ii), (9)(ii), and (10)(ii).13. The at least one nucleic acid according to embodiment 12, wherein each additional CH1 domain matches an additional CL domain.14. The at least one nucleic acid according to embodiment 12 or 13, wherein an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain.15. The at least one nucleic acid according to any one of embodiments 12 to 14, wherein the encoded antibody or fragment thereof is selected from the group consisting of an asymmetric IgG antibody and a F(ab’)2 fragment.16. The at least one nucleic acid according to any one of embodiments 12 to 14, wherein the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C- terminus of the antibody or fragment thereof.17. The at least one nucleic acid according to embodiment 12 or 16, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) asymmetric IgG antibody-peptide and F(ab’)2-peptide.18. The at least one nucleic acid according to embodiment 16 or 17, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.19. The at least one nucleic acid according to any one of embodiments 12 or 16 to 18, wherein the fusion protein is selected from the group consisting of asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, F(ab’)2-cytokine2, F(ab’)2- SCFV2, and F(ab’)2-VHH2.20. The at least one nucleic acid according to any one of embodiments 12 to 19, wherein the encoded antibody or fragment thereof or fusion protein comprises two different CH1 domains and two different CL domains.21 . The at least one nucleic acid according to any one of embodiments 1 to 11 , wherein the at least one nucleic acid encodes at least two antibodies or fragments thereof, or fusion proteins comprising the antibody or fragment thereof, wherein each CH1 domain and each CL domain of an encoded first antibody or fragment thereof or fusion protein is selected from the group consisting of (1)(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each CH1 domain and each CL domain of each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain different from (1 )(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and a CL domain different from (1 )(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i).22. The at least one nucleic acid according to embodiment 21 , wherein each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additionally encoded antibody or fragment thereof or fusion protein.23. The at least one nucleic acid according to embodiment 21 or 22, wherein an additionally encoded antibody or fragment thereof or fusion protein comprises a wild-type CH1 domain and a wild-type CL domain.24. The at least one nucleic acid according to any one of embodiments 21 to 23, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an IgG antibody or IgG antibody fragment thereof.25. The at least one nucleic acid according to any one of embodiments 21 to 24, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an lgG1 antibody or IgG 1 antibody fragment thereof.26. The at least one nucleic acid according to any one of embodiments 21 to 25, wherein the CL domain of the at least one additionally encoded antibody or fragment thereof or fusion protein is of kappa isotype.27. The at least one nucleic acid according to any one of embodiments 21 to 26, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an antibody selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.28. The at least one nucleic acid according to any one of embodiments 21 to 26, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.The at least one nucleic acid according to any one of embodiments 21 to 26, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is a fusion protein, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof. The at least one nucleic acid according to embodiment 29, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody-peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody- peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab- peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptidei-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL- peptide-1-2 (preferably peptide2-CH1-CL-peptide2) . The at least one nucleic acid according to embodiment 29 or 30, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH. The at least one nucleic acid according to any one of embodiment 21 to 26 and 29 to 31 , wherein the fusion protein is selected from the group consisting of symmetric IgG antibody- cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody-cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab- cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2- VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1- CL-VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2-CH1-CL-scFv2, VHH2-CHI-CL-VHH2, scFv2-CH1-CL-VHH2and VHH2-CH1-CL-scFv2. The at least one nucleic acid according to any one of embodiments 1 to 32, wherein at least a CH 1 domain is encoded by a first nucleic acid and at least a CL domain is encoded by a second nucleic acid. The at least one nucleic acid according to any one of embodiments 1 to 33, wherein the nucleic acid is DNA or RNA, preferably mRNA. A particle comprising the isolated nucleic acid according to any one of embodiments 1 to 34. The particle according to embodiment 35, wherein the particle is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a baculoviral vector, an Epstein-Barr viral vector, a poxvirus vector, a virosome, a lipid nanoparticle (LNP), a liposome, a lipoplex (LPX), and a polyplex (LPX). A composition comprising the at least one nucleic acid according to any one of embodiments 1 to 34 or the particle according to embodiment 35 or 36.38. The composition according to embodiment 37, wherein the composition is a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.39. The composition according to embodiment 37 or 38 for use in therapy.40. The composition according to embodiment 37 or 38 for use in the treatment of cancer.41. The composition according to embodiment 37 or 38 for use in the treatment of an infectious disease.42. The composition according to embodiment 37 or 38 for use in the treatment of an autoimmune disease.43. An in vitro method of determining the percent of correctly paired heavy and light chains amongst paired heavy and light chains derived from at least two antibodies directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising at least (i) a heavy chain and a light chain of a first antibody and (ii) a light chain of a second antibody; b) determining the total amount of antibody by staining the paired heavy and light chains with an anti-Fc antibody and quantifying the signal of the anti-Fc antibody to arrive at a quantity 1 ; c) determining the amount of the first antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the first antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the first antibody, and quantifying the signal of said anti-idiotype antibody to arrive at a quantity 2a; d) calculating a result according to the formula [quantity 2a I quantity 1] x 100; wherein the result indicates the percent of correctly paired heavy and light chains.44. The method according to embodiment 44, wherein (i) in step a) a light chain and a heavy chain of the second antibody are provided; (ii) the method comprises an additional step c1) of determining the amount of the second antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the second antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the second antibody, and quantifying the signal of said anti-idiotype antibody to arrive at a quantity 2b; and (iii) step d) comprises calculating a result according to the formula [(quantity 2a + quantity 2b) I quantity 1] x 100.45. The method according to embodiment 43 or 44, wherein the paired heavy and light chains are provided in step a) by expressing at least one nucleic acid encoding at least the heavy and the light chain of the first antibody and the light chain of the second antibody in a cell to obtain paired heavy and light chains.46. The method according to any one of embodiments 43 to 45, wherein the paired heavy and light chains are comprised in a solution.47. The method according to embodiment 45 or 46, wherein the obtained paired heavy and light chains are comprised in a cell culture supernatant, optionally wherein the paired heavy and light chains are purified from the cell culture supernatant and / or concentrated.48. The method according to any one of embodiments 45 to 47, wherein the at least one nucleic acid is expressed by transfecting the cell with the at least one nucleic acid, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the at least one nucleic acid is expressed transiently and / or stably.49. The method according to any one of embodiments 43 to 48, wherein the determination steps b), c) and c1) are carried out using an enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA.50. The method according to embodiment 49, wherein the determination step b) is carried out using an anchoring moiety-coupled, preferably biotinylated, anti-human IgG-Fc antibody as capture reagent and a reporter moiety-coupled, preferably fluorophore-coupled, anti-human IgG-Fcy antibody as detection agent.51. The method according to embodiment 49 or 50, wherein the determination step c) is carried out using an anchoring moiety-coupled, preferably biotinylated, anti-human IgG-Fc antibody as capture reagent and a reporter moiety-coupled, preferably fluorophore-coupled, anti-idiotype antibody directed to the first antibody as detection agent.52. The method according to any one of embodiments 49 to 51 , wherein the determination step c1) is carried out using an anchoring moiety-coupled, preferably biotinylated, anti-human IgG- Fc antibody as capture reagent and a reporter moiety-coupled, preferably fluorophore-coupled, anti-idiotype antibody directed to the second antibody as detection agent.53. An in vitro method of detecting mispaired heavy and light chains amongst paired heavy and light chains derived from at least one IgG antibody and at least one Fab fragment directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising (i) a heavy chain and a light chain ofan IgG antibody, wherein the heavy chain is not fused to a tag and the light chain is fused to a tag1 , and (ii) a VH-CH1 -fusion chain and a light chain of a Fab fragment, wherein the VH-CH1 -fusion chain is not fused to a tag and the light chain is fused to a tag2; b) denaturing the paired heavy and light chains provided in step a) under nonreducing conditions; c) separating the denatured paired heavy and light chains according to the molecular weight; and d) staining the paired heavy and light chains separated according to molecular weight with (i) a detection agent specific for tag1 , (ii) a detection agent specific for tag2; and optionally (iii) a detection agent specific for human Fd; wherein the staining of the different detection agents can be discriminated from each other; wherein tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is such that a discrimination between tag1 and tag2 is possible when separated according to the molecular weight; and wherein mispaired heavy and light chains are identified via their molecular weight and stain; and optionally wherein the ratio of (i) mispaired heavy and light chains to (ii) correctly paired heavy and light chains and single chains is identified via the Fd staining.54. The method according to embodiment 53, wherein (i) paired heavy and light chains of a molecular weight of about (50 kDa + MW1 ) stained with the agent specific for tag1 ; (ii) paired heavy and light chains of about (75 kDa + MW2) stained with the agent specific for tag2; (iii) paired heavy and light chains of about (150 kDa + MW1 + MW2) stained with the agent specific for tag1 and the agent specific for tag2; and (iv) paired heavy and light chains of about (150 kDa + MW2 + MW2) stained with the agent specific for tag2 correspond to mispaired antibody chains.55. The method according to embodiment 53 or 54, wherein the paired heavy and light chains are provided in step a) by expressing at least one nucleic acid encoding the chains in a cell to obtain paired heavy and light chains.56. The method according to any one of embodiments 53 to 55, wherein the paired heavy and light chains are comprised in a solution.57. The method according to embodiment 55 or 56, wherein the obtained paired heavy and light chains are comprised in a cell culture supernatant, optionally wherein the paired heavy and light chains are purified from the cell culture supernatant and / or concentrated.58. The method according to any one of embodiments 55 to 57, wherein the at least one nucleic acid is expressed by transfecting the cell with the at least one nucleic acid, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the at least one nucleic acid is expressed transiently and / or stably.59. The method according to any one of embodiments 53 to 58, wherein tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is at least about 3 kDa, preferably at least about 5 kDa, more preferably at least about 10 kDa and most preferably at least about 15 kDa.60. The method according to any one of embodiments 53 to 59, wherein tag1 and / or tag2 is / are fused to the N-terminus of the chain.61 . The method according to any one of embodiments 53 to 60, wherein the denaturing in step b) is carried out using a detergent, preferably SDS, and / or heat.62. The method according to any one of embodiments 53 to 61 , wherein step c) is carried out by a polyacrylamide gel electrophoresis (PAGE), preferably an SDS-PAGE.63. The method according to any one of embodiments 53 to 62, wherein step d) is carried out by blotting and incubating with the detection agents, preferably a Western Blot.64. The method according to any of embodiments 53 to 63, wherein steps c) to d) are carried out by an SDS-PAGE followed by a Western-Blot.65. The method according to any one of embodiments 53 to 64, wherein the detection agent specific for human Fd is an anti-human Fd antibody coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme and wherein the stain is preferably a fluorescent or luminescent signal.66. The method according to any one of embodiments 53 to 65, wherein the detection agent specific for tag1 is an antibody directed to tag1 coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme and wherein the stain is preferably a fluorescent or luminescent signal.67. The method according to any one of embodiments 53 to 66, wherein the detection agent specific for tag2 is an antibody directed to tag2 coupled to a reporter moiety, wherein the reporter moiety is preferably a fluorophore or an enzyme and wherein the stain is preferably a fluorescent or luminescent signal.68. The method according to any one of embodiments 53 to 67, wherein tag1 and tag2 are selectedfrom the group consisting of a FLAG tag, a His-tag, a RGS-His-GpL tag, a GST tag, an MBP tag, a SUMO tag, a GFP tag, a TAP tag, a TRX tag, an HA tag, a Myc tag, a V5 tag, and a CBP tag.69. The method according to any one of embodiments 53 to 68, wherein tag1 is a FLAG-tag.70. The method according to any one of embodiments 53 to 69, wherein tag2 is an RGS-His-GpL tag.

[0209] Additionally, the present invention relates to the following numbered embodiments:1. At least one nucleic acid encoding at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU;(2)(i) a CH1 domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or (i”) G141 W and K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’”) A125M, S127I and V190Win the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU, and (2)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU;(3)(i) a CH1 domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU, and (3)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU; and(4)(i) a CH1 domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (4)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU.The at least one nucleic acid according to embodiment 1 , wherein the at least one nucleic acid encodes an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the CH1 domain and the CL domain, or a fusion protein comprising said antibody or fragment thereof, wherein the antibody is preferably selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody; wherein the fragment is preferably selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment; and the fusion protein is preferably selected from the group consisting of symmetric IgG antibody- peptide2, asymmetric IgG antibody-peptide2, Fab-peptide2, F(ab’)2-peptide2, peptide2-CH1-CL, preferably CH1-CL-peptide2 and peptide2-CH1-CL-peptide2 with the peptide being selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH. The at least one nucleic acid according to embodiment 1 or 2, wherein the at least one nucleic acid encodes an antibody or fragment thereof, or a fusion protein comprising said antibody or fragment thereof, wherein the antibody or fragment thereof comprises at least two different CH 1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are selected from the group consisting of (1)(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), and (4)(i) and (4)(ii); and wherein each additional CH1 domain comprises a CH1 domain different from (1 )(i), (2)(i), (3)(i) and (4)(i), and each additional CL domain comprises a CL domain different from (1)(ii), (2)(ii), (3)(ii), and (4)(ii). The at least one nucleic acid according to embodiment 3, wherein each additional CH1 domain matches an additional CL domain, optionally wherein an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain, and preferably wherein the encoded antibody or fragment thereof or fusion protein comprises two different CH1 domains and two different CL domains. The at least one nucleic acid according to embodiment 1 or 2, wherein the at least one nucleic acid encodes at least two antibodies or fragments thereof, or fusion proteins comprising the antibody or fragment thereof, wherein each CH1 domain and each CL domain of an encoded first antibody or fragment thereof or fusion protein is selected from the group consisting of (1 )(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), and (4)(i) and (4)(ii); and wherein each CH1 domain and each CL domain of each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain different from (1 )(i), (2)(i), (3)(i) and (4)(i), and a CL domain different from (1)(ii), (2)(ii), (3)(ii), and (4)(ii). The at least one nucleic acid according to embodiment 5, wherein each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additionally encoded antibody or fragment thereof or fusion protein, optionally wherein an additionally encoded antibody or fragment thereof or fusion protein comprises a wild-type CH1 domain and a wild-type CL domain.7. The at least one nucleic acid according to any one of embodiments 1 to 6, wherein at least a CH 1 domain is encoded by a first nucleic acid and at least a CL domain is encoded by a second nucleic acid, wherein the nucleic acid is preferably mRNA.8. A particle comprising the isolated nucleic acid according to any one of embodiments 1 to 7.9. The particle according to embodiment 8, wherein the particle is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a baculoviral vector, an Epstein-Barr viral vector, a poxvirus vector, a virosome, a lipid nanoparticle (LNP), a liposome, a lipoplex (LPX), and a polyplex (LPX).10. A composition comprising the at least one nucleic acid according to any one of embodiments 1 to 7 or the particle according to embodiment 8 or 9, preferably wherein the composition is a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.11. The composition according to embodiment 10 for use in therapy, preferably for use in the treatment of cancer or an infectious disease or an autoimmune disease.12. An in vitro method of determining the percent of correctly paired heavy and light chains amongst paired heavy and light chains derived from at least two antibodies directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising at least (i) a heavy chain and a light chain of a first antibody and (ii) a light chain of a second antibody; b) determining the total amount of antibody by staining the paired heavy and light chains with an anti-Fc antibody and quantifying the signal of the anti-Fc antibody to arrive at a quantity 1 ; c) determining the amount of the first antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the first antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the first antibody, and quantifying the signal of said anti-idiotype antibody to arrive at a quantity 2a; d) calculating a result according to the formula [quantity 2a / quantity 1] x 100; wherein the result indicates the percent of correctly paired heavy and light chains.13. The method according to embodiment 12, wherein the determination steps b) and c) are carriedout using an enzyme-linked immunosorbent assay (ELISA), preferably (i) wherein the determination step b) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety-coupled coupled anti-human IgG-Fcy antibody as detection agent; and (ii) the determination step c) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reported moiety-coupled anti-idiotype antibody directed to the first antibody as detection agent. An in vitro method of detecting mispaired heavy and light chains amongst paired heavy and light chains derived from at least one IgG antibody and at least one Fab fragment directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising (i) a heavy chain and a light chain of an IgG antibody, wherein the heavy chain is not fused to a tag and the light chain is fused to a tag1 , and (ii) a VH-CH1-fusion chain and a light chain of a Fab fragment, wherein the VH-CH1 -fusion chain is not fused to a tag and the light chain is fused to a tag2; b) denaturing the paired heavy and light chains provided in step a) under nonreducing conditions; c) separating the denatured paired heavy and light chains according to the molecular weight; and d) staining the paired heavy and light chains separated according to molecular weight with (i) a detection agent specific for tag1 , (ii) a detection agent specific for tag2; and optionally (iii) a detection agent specific for human Fd; wherein the staining of the different detection agents can be discriminated from each other; wherein tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is such that a discrimination between tag1 and tag2 is possible when separated according to the molecular weight; and wherein mispaired heavy and light chains are identified via their molecular weight and stain; and optionally wherein the ratio of (i) mispaired heavy and light chains to (ii) correctly paired heavy and light chains and single chains is identified via the Fd staining. The method according to embodiment 14, wherein (i) paired heavy and light chains of a molecular weight of about (50 kDa + MW1 ) stained with the agent specific for tag1 ; (ii) paired heavy and light chains of about (75 kDa + MW2) stained with the agent specific for tag2; (iii) paired heavy and light chains of about (150 kDa + MW1 + MW2) stained with the agent specific for tag1 and the agent specific for tag2; and (iv) paired heavy and light chains of about (150kDa + MW2 + MW2) stained with the agent specific for tag2 correspond to mispaired antibody chains.Sequences

[0210] It is evident for the skilled person that the nucleotide sequences depend on the individual context for cloning and codon-optimization as described herein but still encode the same amino acid sequence. Examples

[0211] The following Examples are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.Example 1 : Establishment of a Western Blot-based method for CH1-CL domain mispairinq assessment

[0212] The present example demonstrates the establishment of a Western Blot-based method for the analysis of CH1 and CL domain mispairing in co-transfection mixes of wildtype chains of different molecular weights by distinguishing between different band sizes.

[0213] In co-transfections of unmodified wildtype IgG and Fab chains, statistically, both the HC chains of the IgG model antibody and the Fab fragment model antibody will pair with the LC chains of both the IgG and Fab fragment. Heavy chain to heavy chain mispairing will not occur. Based on the different antibody formats used, as well as the additional RGS-His tag with molecular weight GpL spacer (18.17 kDa) at the N-terminus of the mutated Fab LC, and the Flag tag (1.01 kDa) at the N-terminus of the wildtype LC, all four produced antibody fragments differed significantly in their molecular weight, allowing for differentiation of the individual chains: Two DNA plasmids encoding a first IgG model antibody 1 were cloned and transiently produced, having molecular weights of 49.40 kDa (HC1 model Ab1) 25.17 kDa (LC1 model Ab1 with Flag- peptide tag). In addition, three DNA plasmids encoding a second model antibody 2 in the Fab format were also cloned and transiently produced, having molecular weights of 23.48 kDa (HC2 model Ab2) and 24.65 kDa (LC2_v2 model Ab2 with RGS-His- peptide tag) or 42.50 kDa (LC2 model Ab2 with RGS-His-GpL-peptide tag) (Fig. 1 and Fig. 2). Cotransfections of HC1 +LC1 +HC2+LC2 and HC1 +LC1 +HC2+LC2_v2 were carried out and IgG and Fab chains secreted into the cell culture supernatant were analyzed by making use of non-reducingWestern Blots. Detection of the individual chain pairings was performed using anti-human IgG FD- specific, anti-His-specific and anti-Flag-specific detection antibodies to distinguish between all different assemblies of correctly and mispaired antibody chains based on different band sizes and peptide tag detection (Fig. 3).

[0214] Methods of the present example include:1.1 Cloning

[0215] For assay establishment, the sequences encoding antibody fragments composed of a) variable heavy and variable light chain domains of two model antibodies (model Ab1 and model Ab2), b) constant domains of the human immunoglobulin 1 isotype and c) selected tags and / or molecular spacer elements (Fig. 1) were synthesized either internally or externally (Thermo Fischer Scientific) and then cloned into the mammalian expression vector pcDNA3.4-TOPO for subsequent expression of the encoded antibody sequences in a mammalian host system. For optimal expression of the antibody in human cells, the heavy- and light chain-encoding sequences were codon optimized. Optimization was carried out with the GeneOptimizer® software provided by Life Technologies GmbH GeneArt®. This software adjusts the codon usage by using the most frequent codons and adapts the GC-content of an uploaded sequence for a chosen expression system, in this case Homo sapiens. The output sequence was not further manipulated and used as it was for analysis and ordering of DNA fragment strings. Cloning was performed by techniques that are commonly known by people skilled in the art. In short, each light chain and heavy chain sequence encoded in the DNA fragment strings was cloned into pcDNA3.4-TOPO via Xbal and Agel restriction sites. For this purpose, the parental pcDNA3.4-TOPO vector was digested with the restriction enzymes Xbal and Agel. The resulting product was analyzed via agarose gel, purified and its concentration measured by UV spectroscopy. The antibody encoding light chain and heavy chain sequences containing complimentary 30bp 5’ and 3’ overhangs homologous to the ends of restriction digested vector and matching the Xbal and Agel adjacent plasmid regions, were assembled with the pcDNA3.4-TOPO vector by the In Vivo Assembly cloning method according to Garcia-Nafria et al. (Garcia-Nafria, “IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly,” Scientific Reports volume 6, Article number: 27459 (2016), which is incorporated herein by reference in its entirety.) In short, digested pcDNA3.4- TOPO vector and antibody encoding sequences were mixed and competent Escherichia coli TOP10 cells (Life Technologies) were transformed via heat shock. Clones were sequence verified by Sanger sequencing. Sequencing results were aligned to the reference sequence. The nucleotide sequence of the resulting heavy chain- and light chain-encoding plasmid constructs can be found in the following sequences and are detailed below in Table 1 .Table 1 : Nucleotide sequences of cloned DNA constructs for antibody production used for CH1-CL domain mispairing assessment in the Western Blot-based method. SP, signal peptide; VH, variable heavy chain domain; VL, variable light chain domain; CH1 , constant heavy chain domain1 ; CH1-3, constant heavy chain domains 1 , hinge, 2 and 3; Ckappa, human constant kappa domain; hlgG1 , human immunoglobulin 1 isotype; His, 6xHis-tag; GpL, Gaussia princeps luciferase-tag.

[0216] Plasmid DNA was prepared by selecting E. coli clones for inoculation in Luria-Bertani (LB) medium containing carbenicillin. The cultures were grown overnight at 37 °C and ~ 150 to 200 rpm. Following cell harvest, purification was done using the QIAGEN Plasmid Plus Maxi Kit (QIAGEN GmbH) according to the manufacturer’s instructions relying on alkaline lysis and subsequent silica column purification. The concentration was determined by UV spectroscopy and the successful selection of each novel plasmid was verified by Sanger Sequencing of the respective insert sequence. Finally, DNA was stored in certified RNase- and DNase-free reaction tubes. An overview of the individual plasmids is shown in Table 2.Table 2: Overview of cloned DNA constructs for antibody production used for CH1-CL domain mispairing assessment in the Western Blot-based method. Vector and insert combinations for the different constructs including the insert size in base pairs (bp) and the identifier “Plasmid #”,SP, signal peptide; VH, variable heavy chain domain; VL, variable light chain domain; CH1 , constant heavy chain domain 1 ; CH1-3, constant heavy chain domains 1 , hinge, 2 and 3; Ckappa, human constant kappa domain; His, 6xHis-tag; GpL, Gaussia princeps luciferase-tag.1.2 Expression of Model Antibody IgG and Fab Construct Mixes via transient Transfection

[0217] HEK 293ExpiF suspension cells were transiently transfected in three biological replicates with a plasmid mix encoding for both the IgG and Fab model antibody heavy and light chains, as shown in Table 3, according to the manufacturers manual for the 293ExpiF expression system (Thermo FischerScientific). Briefly, the indicated plasmid DNAs encoding the different model antibodies' heavy and light chains were mixed in the indicated combination and chain ratio in Opti-MEM™ I Medium (Gibco). Then ExpiFectamine™ 293 Reagent was mixed with the plasmid DNA swirling or inversion and transferred to the production cell line after complexation. The culture volume was 500 pL for each transfection in 2 mL 96 deep well plates (Carl Roth) sealed with a gas permeable adhesive seal (LifeTechnologies). Cultivation was conducted for either four or five days shaking at 250 rpm with 50 mm Amplitude, 37°C, 8% CO2 and >80% humidity. The cells were harvested via centrifugation twice at 1500xg to obtain cell culture supernatant for subsequent analysis. Table 3: DNA chain mixes for model antibody production via transient transfection in Example1. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of correctly assembled wildtype IgG chains (DNA-1 +DNA-2) or Fab chains (DNA-3+DNA-4 or DNA-3+DNA-5) aswell as for mispairing analysis of co-transfected IgG and Fab chains (DNA-1+DNA-2+DNA-3+DNA-4 or DNA-1+DNA-2+DNA-3+DNA-5).1.3 Gyros ELISA for Model Antibody IgG and Fab Constructs Quantitation

[0218] Total human IgG and Fab model antibody concentrations in cell culture supernatant mixes was determined via a sandwich immunoassay using the Gyros xPand™ XPA1055 immunoassay system (GyroLab). The assays were conducted in a Bioaffy CD20HC disc (GyroLab) with the huIgG High Titer Kit (GyroLab) using a biotinylated goat F(ab’)2 fragment anti-human lgG(H+L) (Jackson ImmunoResearch Europe Ltd) as capture antibody and a AlexaFluor647 conjugated goat IgG antihuman F(ab’)2 fragment specific (Jackson ImmunoResearch Europe Ltd) detection antibody. A standard curve was prepared with a previously purified batch of the model IgG antibody and the antibody concentration in the cell culture supernatant was calculated using the GyroLab Evaluator software.1.4 Western Blot Analysis of Model Antibody IgG and Fab constructs in Cell Culture Supernatants

[0219] The chain pairings of the lgG1 and Fab model antibody mixes within the cell culture supernatant samples were assessed via Western Blots under non-reducing conditions with an HRP coupled goat anti-human IgG FD detection antibody (1 :1000) (Cell Sciences / Holzel Diagnostika Hand.) according to standard procedures well known by the person skilled in the art. In short, the proteins were separated via polyacrylamide gel electrophoresis using a 4-15% Criterion™ TGX Stain- Free™ Gel (Bio-Rad Laboratories, Dreieich, Germany). HRP was detected using the Clarity Western ECL substrate (Bio-Rad Laboratories) and the luminescence recorded with a Chemidoc MP Imaging System (Bio-Rad Laboratories). Calculation of the Western Blot band luminescence intensities was carried out using the ImageLab software (Bio-Rad Laboratories).

[0220] In addition, for verification of identified molecular weight species, Western Blot staining was also performed under non-reducing conditions using fluorescence-coupled anti-peptide tag antibodies. Here, the RGS-His-tag was detected with an Alexa Fluor® 488-coupled mouse anti-His Tag antibody (BioLegend) and the Flag-Tag was detected with an Alexa Fluor® 647-coupled rat anti-DYKDDDDK Tag antibody (Clone L5; Invitrogen), allowing to distinguish between band signals of pairings containing either His- or Flag-tagged light chains or both (Fig. 3A).

[0221] Apart from detection of assembled antibody chains, the production and integrity of the individual chains was also checked under reducing Western Blot conditions using Dithiothreitol (DTT) in combination with the detection antibodies and conditions described above (Fig. 3B).1.5 Results

[0222] Table 4 provides a summary of the IgG and Fab model antibody chain pairings that are theoretically possible after co-transfections of either HC1+LC1+HC2+LC2 or HC1+LC1+HC2+LC2_v2. It can be assumed that different complete and incomplete assemblies are possibly secreted during model antibody production, which can result in correct chain pairings (square HC + square LC or triangle HC + triangle LC), but also in mispairings (square + triangle).

[0223] The results from the Western Blot analysis are shown in Fig. 3. Under reducing conditions, the production of all individual chains was confirmed, and the light chains were also selectively detected via the respective peptide tags (Fig. 3B). Under non-reducing conditions, the expected variety of occurring chain pairings became visible using the different detection antibodies (Fig. 3A). Here, the IgG and Fab model antibody mix produced from DNA-1 / -2 / -3 / -5, including the LC2_v2 with RGS-His peptide-tag, resulted in a single band at the expected size of the fully assembled IgG monomer (approx. 150 kDa) using the anti-Flag peptide tag detection antibody and a band at the same size using the anti-His peptide tag detection antibody. This proves the existence of at least two species at this size, containing both correctly assembled model antibody 1 (HC1+LC1) as well as mispaired model antibody 1 / 2 (HC1+LC2) species. Following detection with an anti-Fd-specific antibody, additional chain pairings were observed using this IgG and Fab model antibody mix produced from DNA-1 / -2 / -3 / -5, indicating that also a mispairing species of model antibody 2 / 1 (HC2+LC1) can be detected at approx. 49 kDa.

[0224] Alternatively, the IgG and Fab model antibody mix produced from DNA-1 / -2 / -3 / -4, including the LC2 with RGS-His-GpL peptide-tag, resulted in a broader band pattern at various expected sizes. Here, using the anti-Flag peptide tag detection antibody, correctly assembled IgG monomer (HC1+LC1+HC1+LC1) can be detected and distinguished from a mispairing species (HC1 +LC1 +HC1 +LC2), that became visible as separate band at approx. 167 kDa. This species was also detected with the anti-His peptide tag detection antibody as well as an additional mispairing species at approx. 184 kDa, representing the HC1+LC2+HC1+LC2 species. Again, following detection with an anti-Fd-specific antibody, all expected chain pairings from Table 4 were observed using this IgG and Fab model antibody mix produced from DNA-1 / -2 / -3 / -4. Here, the correctly assembled antibody species are distinguishable from the mispaired species by different band sizes using this single detection antibody. Hence, it was concluded that the Western Blot assay was successfullyestablished with the DNA-1 / -2 / -3 / -4 mix, allowing for detection of the individual IgG and Fab model antibody chains as well as differentiation of the correctly assembled from the mispaired species. This allowed for further application of this method in the assessment of CH1-CL mispairing prevention mutations, as it can be assumed that less mispairing will occur between the IgG and Fab chains with beneficial novel mutations being implemented in the CH1 and CL domains of the Fab fragment, resulting in fewer or weaker bands detectable at different band sizes.Table 4: Overview of possible chain pairings after transient co-transfections of the indicated chainsExample 2: Screening of CH1-CL mutation pairs for antibody productivity and CH1-CL mispairinq proportions via Western Blot-based method

[0225] The present example shows the results of the screening of candidate mutation pairs for CH 1- CL mispairing prevention. For the comparison of the effects of the different mutations on antibody mispairing, the established Western Blot-based assay described in Example 1 has been utilized. To avoid heavy chain to heavy chain mispairing the assay is conducted using a wildtype IgG model Ab1 and a second model Ab2 expressed as a Fab. In the Fab model Ab sequence the different CH1 and CL mutations were introduced via cloning, resulting in the plasmid constructs according to Table 5 with the CH1 and CL domain sequences shown in Table 6. With the plasmid ratios described in Table 3, the IgG / Fab mixes were expressed in parallel in HEK 293ExpiF cells via transient transfection. The resulting supernatants were analyzed for antibody titers via automated ELISA to assess differences in expression compared to the wildtype Fab or literature controls. To compare mispairing between the candidates, equal amounts of each co-transfection mix were analyzed on non-reduced Western Blots according to the different molecular weights of each chain pairing (Fig. 6). In addition, the different resulting Western Blot band signal intensities were determined via image analysis. The ratio of the value of the additional bands from mispairing and that of all detected bands, except those species that missed light chains, was calculated to obtain the mispairing values.

[0226] Methods of the present example include:2.1 Cloning

[0227] Cloning was performed by techniques that are commonly known by people skilled in the art. In short, each light chain and heavy chain sequence encoded in the DNA fragment strings was cloned into pcDNA3.4-TOPO via Xbal and Agel restriction sites. For this purpose, the parental pcDNA3.4- TOPO vector was digested with the restriction enzymes Xbal and Agel. The resulting product was analyzed via agarose gel electrophoresis, purified and its concentration measured by UV spectroscopy. After selection of preferred mutations for screening, gene syntheses coding for the different CH 1 and CL variants were generated either internally or externally (Thermo Fischer Scientific) with added 30bp overhangs on each end homologous to the ends of the previously restriction digested vectors. To assemble the expression vector pCDNA3.4-TOPO with the gene synthesis the E. coli Top10 strain and the In Vivo Assembly cloning method according to Garcia-Nafria et al. (Garcia-Nafria, “IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly,” Scientific Reports volume 6, Article number: 27459 (2016), which is incorporated herein by reference in its entirety.) were utilized. After transformation clones were selected using LB-Agar plates with 100 pg / mL Carbenicillin added and the resulting clones sequence verified using Sanger sequencing. The resulting plasmids are described in Table 5. Large scale plasmid preparations were generated using 50 mL E. coli overnight cultures in LB-Carbenicillin medium and the Plasmid Plus Midi Kit (QIAGEN GmbH) relying on alkaline lysis and subsequent silica column purification. The plasmid concentrations were determined using UV spectroscopy. The other candidate sequence plasmids were generated in an analogous manner. Due to the initial parental sequences of model Ab1 and model Ab2, different CH1 allotypes (G1 m3 and G1 m17) were used for cloning of the plasmids DNA-1 to DNA-21 , resulting in a difference of one amino acid in the very C-terminus of the CH1 domains that was considered uncriticalfor assessment of CH1-CL mispairing during the Western Blot-based assay. For cloning of plasmids DNA-22 to DNA-60 (Table 7) and use in the ELISA-based mispairing assay (Example 3), the G1 m3 allotype was uniformly used. Table 5: Overview of cloned DNA constructs for antibody production used for screening of CH1-CL domain mutation pairs. Vector and insert combinations for the different constructs including the insert size in base pairs (bp) and the identifier “PlasmidSP, signal peptide; VH, variable heavy chain domain; VL, variable light chain domain; CH1 , constant heavy chain domain 1 ; CH1-3, constant heavy chain domains 1 , hinge, 2 and 3; Ckappa, human constant kappa domain;; His, 6xHis-tag; GpL, Gaussia princeps luciferase-tag; model Ab1 , monoclonal model antibody 1 ; model Ab2, monoclonal model antibody 2. Names of CH1-CL domain mutations: e.g. Leadl . CV refers to the charge variants of the CrossMab technology, DM refers to the disulfide bond replacement mutations of the DuetMab technology.2.2 Production

[0228] HEK 293ExpiF cells were transiently transfected in three biological replicates according to the manufacturers manual for the 293ExpiF expression system (Thermo Fischer Scientific). For the transfection, a plasmid mix was used consisting of chains for both the IgG and Fab model antibody heavy and light chains, according to the ratios shown in Table 3 with the controls being the same and for the individual candidates only the respective Fab-HC and Fab-LC was exchanged. The inserted CH1 and CL domain sequences are shown in Table 6. The culture volume was 500 pL for each transfection in 2 mL 96 deep well plates (Carl Roth) sealed with a gas permeable adhesive seal (Life Technologies). Cultivation was conducted for either four or five days shaking at 250 rpm with 50 mm Amplitude, 37°C, 8% CO2 and >80% humidity. The cells were harvested via centrifugation twice at 1500xg to obtain cell culture supernatant for subsequent analysis.Table 6: Overview of CH1 and CL domain sequences of lead mutations and compared technologies. The sequence describes only the optimized DNA sequence coding for the altered CH1 or CL domain respectively. CH1 , human lgG1 constant heavy chain domain 1 ; Ckappa, human constant kappa domain; CrossMab refers to the charge variants of the CrossMab technology, DuetMab refers to the disulfide bond replacement mutations of the same name.2.3 Antibody Expression Analysis

[0229] Total human lgG1 and Fab model Ab mix concentration was determined via sandwich immunoassay using the Gyros xPand™ XPA1055 immunoassay system (GyroLab). The assays were conducted in a Bioaffy CD20HC disc (GyroLab) with the huIgG Low or High Titer Kit (GyroLab) respectively using a biotinylated goat F(ab’)2 fragment anti-human lgG(H+L) (Jackson ImmunoResearch Europe Ltd) as capture antibody and a AlexaFluor647 conjugated goat IgG antihuman F(ab’)2 fragment specific (Jackson ImmunoResearch Europe Ltd) detection antibody. The standard curve was prepared with the purified model IgG antibody diluted in transfection mock supernatant and the results calculated using the GyroLab Evaluator software.2.4 Western Blot CH1-CL Mispairing Analysis

[0230] The comparative CH1-CL mispairing of cell culture supernatant samples containing the expressed model Ab1 and model Ab2 mixes was assessed via Western Blot under non-reducing conditions with an HRP coupled goat anti-human IgG FD detection antibody (Cellsciences / Holzel Diagnostika Hand.) according to standard procedures well known by the person skilled in the art. The proteins were applied in equal amounts based on the previous ELISA results and separated via polyacrylamide gel electrophoresis using a 4-15% Criterion™ TGX Stain-Free™ Gel (Bio-Rad Laboratories, Dreieich, Germany). HRP was detected using the Clarity Western ECL substrate (BioRad Laboratories) and the luminescence recorded with a Chemidoc MP Imaging System (Bio-Rad Laboratories). Calculation of the Western Blot band luminescence intensities was carried out using the ImageLab software (Bio-Rad Laboratories). The adjusted false pairing was calculated by determining the percentage ratio of the Western Blot band signal intensities of the incorrectly paired antibody species to all antibody species on the Western Blot. Excluded were those antibody species that did not show a heavy and light chain pairing, neither correct nor mispaired, like individual chains and heavy chain only dimers. Furthermore, the antibody species with two heavy chains of the full IgG antibody and only one light chain of the Fab antibody was excluded because of its low ratio, rarely over the Western Blot detection limit, and its proximity to the intact full IgG band.2.5 Results

[0231] Both the expression analysis via ELISA and the mispairing analysis via Western Blot were conducted in parallel with several controls. “Control CrossMab” (CV) and “Control DuetMab” (DM), which were mixes including the CrossMab and DuetMab mutations and were used to compare novel mispairing prevention candidates to common technologies used in the literature (WO2015150447A1 , WO2017055539A1 and WO2013096291A2). The “Control Fab” and “Control IgG” samples were generated by only transfecting the cells with DNA coding for either IgG or Fab model antibody and were used to illustrate which bands the correctly paired antibody species produce in the Western Blot. The “Control IgG / Fab” sample was produced using both IgG model Ab1 and wildtype Fab model Ab2 sequences and served as a control for the statistical mispairing of wildtype CH1-CL domains expressed simultaneously in a cell and for the expression that could be expected from the two model antibodies as a mix. The expression analysis via ELISA showed that most mixes with novel mispairing prevention candidates including the leads yielded titers between 2 to 6 pg / mL (Fig. 4), indicating no apparent differences to literature controls. Differences to the mix “Control IgG / Fab” can be attributed to variations not uncommon with transfection, expression and cultivation of cell cultures in the 96-well format. This is supported by the results seen for the screening of the additional candidates (Fig. 5), where the titer of the “Control IgG / Fab” sample is in the same range as the candidates. Only a few of the candidates, but none of the leads, exhibit changes in expression titers to below 1 .5 pg / mL possibly due to the introduction of mutations in the CH1 and CL domains (Fig. 4). Concerning the additional mutations, tested in a separate assay (Fig. 5), two candidates showed almost no expression of model antibodies in the supernatant and were thus neglected for further analysis, while the others including the leads performed at least comparable than the literature controls for CrossMab, the DuetMab and the “Control IgG / Fab” sample. For the Western Blot mispairing assay, equal masses of total antibody mix for all samples were applied and detected on different Western Blots (Fig. 6A / B) For comparison, the signal intensities of the different antibody species bands on the Western Blot were determined, whose identity had been confirmed in the assay establishment in Example 1. The two uppermost bands correspond to mispaired variants of the IgG, with either one or two light chains of the Fab model antibody. The most intense band in the Western Blot in all samples is the correctly paired IgG model antibody. The two bands below the full IgG are IgG species that either miss one or two light chains altogether. Lastly, the band at around 60 kDa shows the correctly paired Fab, while the band at around 40 kDa is that of the mispaired Fab, with the faint band underneath being free light chain. For all samples on the first Western Blot (Fig .6A) less mispaired antibody species than for the “Control IgG / Fab” sample could be detected, indicating an improvement compared to the statistical mispairing of the wildtype sequences. In case of the second Westen Blot (Fig. 6B) only the lead sequences showed an improvement compared to the “Control IgG / Fab” samples. In several Western Blots, the six lead mutation pairs showed the lowest ratio of mispaired species bands. The signals of the mispaired species bands detected is in the range of that of the CrossMab and DuetMab samples on the same Western Blot respectively, which is especially low for Lead4, Lead5 and Lead6. Consequently, the lead candidates 1-6 were chosen for more detailed analyses including a quantitative mispairing assay based on model antibody binding anti-idiotype antibodies.Example 3: Assessment of antibody domain mispairinq proportions of selected novel sets of CH 1 -CL mutation pairs via ELISA-based method

[0232] The present example shows the results of the assessment of antibody domain mispairing proportions after co-transfection of a mixture of four DNA plasmids encoding two different full IgG model antibodies with either wildtype or mutated CH1 and CL domains. For this purpose, novel plasmids encoding the CH1 and CL sequence with the mispairing prevention mutations, were generated and produced in mixes of wt-HC1 +wt-LC1+neo-HC2+neo-LC2 or wt-HC2+wt-LC2+neo- HC1 +neo-LC1 via transient co-transfection along with control mixes. IgG chains and assembled model Ab1 and model Ab3 species secreted into the cell culture supernatant were analyzed by making use of a sandwich immunoassay (GyroLab ELISA). First, the total antibody concentration of heavy chain containing species was determined using anti-Fc detection antibodies. Detection of HC1 / LC1 and HC2 / LC2 chain pairings was performed using individual anti-ldiotype antibodies directed against either model Ab1 or model Ab3 and capable of exclusive detection of correctly paired individual Fab domains (Fig. 7). Finally, the proportion of correctly paired Fab domains was calculated by dividing the sum of both individual antibody concentrations measured via anti-idiotype antibodies by the total antibody concentration.

[0233] Methods of the present example include:3.1 Cloning

[0234] Plasmids were ordered externally (Thermo Fischer Scientific, Twist Bioscience). The plasmids contained a backbone for expression in mammalian cells (pcDNA3.4-TOPO or pTwist CMV) and an insert encoding for either model Ab1 or model Ab3 fused to either the wildtype CH1 and CL sequence or the CH1 and CL sequences with mispairing prevention mutations in combination with the Fc domains to generate full IgG antibody species (Table 7). Cloning and production of the different DNA plasmids was performed according to the previously described methods (see 1.1 and 2.1 above).Table 7: Overview of cloned DNA constructs for antibody production used for anti-idiotype ELISA-based method for CH1-CL domain mispairing assessment. Vector and insert combinations for the different constructs including the insert size in base pairs (bp) and the identifier “Plasmid SP, signal peptide; VH, variable heavy chain domain; VL, variable light chain domain; CH1 , constant heavy chain domain 1 ; CH1-3, constant heavy chain domains 1 , hinge, 2 and 3; Ckappa, human constant kappa domain; hlgG1 , human immunoglobulin 1 isotype; model Ab1 , monoclonal model antibody 1 ; model Ab3, monoclonal model antibody 3.3.2 Expression of Model Antibody IgG Construct Mixes via transient Transfection

[0235] Expression of IgG model Ab1 and model Ab3 chain mixes was performed via transient transfection with a plasmid mix encoding for the four IgG model antibody heavy and light chains in three biological replicates, as shown in Table 8, according to the manufacturers manual for the 293ExpiF expression system according to the previously described methods (see 1 .2 and 2.3 above).Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers from the different plasmid backbones to prevent biases in the assay from missing or underrepresented chains. Cell culture supernatant containing the differently assembled antibody species was collected 4 or 5 days after transfection and directly used for further Gyros ELISA analysis.Table 8: DNA chain mixes used for antibody production via transient transfection in Example 3.Plasmid mixes and their respective chain mass ratio used for transient co-transfection of correctly assembled wildtype IgG chains (DNA-1+DNA-22 and DNA-39+DNA-40) as well as for mispairing analysis of co-transfected IgG chains (DNA-1+DNA-40 or DNA-22+DNA-39), resulting in CCSN with 100% mispaired model antibody species. Additionally, the last three rows show mixes of DNA chains encoding for four different model antibody chains either carrying wildtype CH1-CL domain-encoding sequences or a mix of wildtype and neo CH1-CL domain-encoding sequences with various mispairing prevention mutations according to the present invention.3.3 Gyros ELISA for Model Antibody IgG and Fab Constructs Quantitation

[0236] The concentrations of correctly paired and mispaired model Ab1 and model Ab3 species were analyzed using the Gyros xPand™ XPA1055 immunoassay system (GyroLab) with a Bioaffy CD20HC disc (GyroLab) and the huIgG Low or High Titer Kit (GyroLab). Total antibody concentration was determined using biotinylated anti-human IgG-Fc antibody (Thermo Fisher Scientific) as the capture reagent and an AlexaFluor647 coupled anti-human IgG-Fcy (Jackson ImmunoResearch Europe Ltd) as the detection reagent. To assess the concentration of the correctly paired individual Fab domains of model Ab1 and model Ab3, externally (Bio-Rad) generated, internally AlexaFluor647-coupled antiidiotype antibodies were used as a detection reagent, while the capture reagent was the biotinylated anti-human IgG-Fc antibody (Thermo Fisher Scientific) used above. The ratio of correctly paired Fab species was calculated by dividing the sum of model Ab1 and model Ab3 concentrations measured via the anti-idiotype antibodies by the total antibody concentration. Standard curves for total antibody concentration were measured with previously produced and purified model Ab1 and model Ab3 separately, to correct for deviations in response signal between the antibodies. The result of the total antibody concentration was the average of the two individual standard curve-based results weighted by the abundance of individual antibody Fabs assessed via anti-idiotype antibodies. Mixtures of antibodies are expected to also contain antibody species lacking one or two light chains or digested antibody fragments that will not be detected either via the anti-idiotype antibody or the anti-Fc antibody, but might be detected by the other, therefore reducing the apparent correct pairing compared to the actual. To account for this deviation, purified antibody mixes with correctly paired and mispaired variants of the two antibodies (HC1+LC1 , HC2+LC2, HC1+LC2, HC2+LC1) were prepared withdifferent levels of mispaired species spiked into the mix. A mispairing standard curve was then measured the same way as described for the mispairing samples (Fig. 8), to elucidate the relation between apparent and actual mispairing values. Furthermore, CH1-CL mutations are introduced into either modelAbl or model Ab3 while the respective other model antibody retained the wildtype CH1 and CL domain sequences. Replicates (n=2x2) of both model antibody mixes were measured to prevent further deviations based on biases in measurement of a mutated individual antibody. As a reference for the novel mutation designs, several point mutations known from the literature (“CrossMab charge variants” (CV), “Duetmab” (DM), “CrossMab domain swap and charge variants” (X / CV)) were co-tested to assess mispairing proportions and have a comparative indication of the effectiveness of mispairing prevention.3.4 Results

[0237] The ELISA-based mispairing assay was used to detect the correctly paired model antibody Fab assemblies in the mix of the four different model antibody chains via anti-idiotype detection and to determine the proportion of correct pairings in relation to the proportion of the mismatched species. Background detection was ruled out using purified control model antibodies consisting of either correctly assembled HC1+LC1 and HC2+LC2 or mispaired HC1 +LC2 and HC2+LC1 (Fig. 7). The ELISA showed signals only for detection with model Ab1 anti-idiotype in the case of the HC1 / LC1 Fab domains and with model Ab3 anti-idiotype only for HC2 / LC2 Fab domains. Signals for the mispaired variants or the other model antibody respectively were under the limit of detection, proving the assay sensitivity to solely detect correctly paired Fab domains. Since the detection of the total antibody concentration was realized in cell culture supernatant samples via Fc-binding antibodies, the measured mispairing had to be correlated with the actual mispairing through freshly prepared mixes of purified antibodies with various increasing ratios of mispaired species added. The occurrence of antibody species in the mix that had an Fc part but not a complete Fab needed for anti-idiotype detection, meant that a 100% correct pairing would never be measured. The mispairing standard curve (Fig. 8) showed a linear correlation of apparent mispairing through measurement and actual mispairing.

[0238] The CH1-CL mispairing prevention capabilities of the six lead mutational pair sequences, listed in Table 9, were determined in three separate experiments with duplicates for each of the model antibodies harbouring the CH1 and CL mutations expressed alongside the wildtype sequence of the other model antibody. These four individual measurements were averaged and additionally a mean of all three experiments was calculated (Fig. 9). Controls with mutations from the DuetMab technology (DM), the CrossMab charge variant mutations (CV), a domain swap variant (X) and a combination of the charge variant mutations and the domain swap used in the CrossMab technology (X / CV) were included as reference for mispairing proportions. The correct pairing of the chains in the wildtype (WT) mix was detected at about 50-60% in all experiments, being similar to the statistically expected 50% with a difference accounting for deviations in mispairing due to VH and VL interaction. The mispaired variants (MP) showed no correct pairing, verifying the absence of background signal within the measurement. All lead candidates and controls convey improved correct assembly of the two modelantibodies compared to model antibody mixes with wildtype sequences. Lead2, Lead4 and Lead6 perform comparable to the DM, X and CV control, with correct pairing of 80 to 90%. Leadl performs best with a mean correct pairing of more than 90% observed over several experiments, being comparable to values obtained with the X / CV control.Table 9: Overview of selected lead candidates and their CH1 and CL domain mutations.Example 4: Assessment of Fab domain pairing via LC-MS analysis after introduction of selected novel sets of CH 1 -CL mispairinq prevention mutations

[0239] The present example shows the results of the CH1 and CL domain mispairing detected via LC-MS mispairing of a mix of two antibodies, one with the wildtype CH1 and CL domain sequences, the other with the mutations of Leadl , Lead, Lead3, control sequences or another wildtype. The samples were generated via transient transfection of HEK cells with the plasmids generated in Example 3. In preparation for the LC-MS analysis, the sample supernatants were purified via affinity chromatography, the antibody concentration determined via UV-spectrophotometry and the Fc domains cut by means of enzyme digestion. Equal amounts of each antibody mix were loaded onto an RP-HPLC column and analyzed in a QTOF mass spectrometer. The LC-MS mass peak signal comparison of the known individual antibody species’ masses allowed for the determination of heavy and light chain or CH1-CL mispairing.

[0240] Methods of the present example include:4.1 Production of Model Antibody IgG Constructs via transient Transfection

[0241] Mispairing evaluation of Leadl , Lead2, Lead3 and controls was conducted by mixing heavy and light chains of the full IgG model Ab1 and model Ab3 antibodies. For each CH1-CL mispairing prevention variant, the corresponding mutations were introduced to either model Ab1 or model Ab3 CH1 and CL domains, while the other antibody had the wildtype CH1 and CL domains of a human kappa IgG 1 antibody. Expression of IgG model Ab1 and model Ab3 chain mixes was performed via transient transfection with a plasmid mix encoding for the four IgG model antibody heavy and lightchains, as shown in Table 10, according to the manufacturers manual for the 293ExpiF expression system (Thermo Scientific) as previously described (see Example 1.2 and 2.2), albeit at 5 mL scale each. Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers from the different plasmid backbones to prevent biases in the assay from missing or underrepresented chains. Samples that showed very low amount of a certain chain were not considered. Cell culture supernatants containing the differently assembled antibody species were collected 4 or 5 days after transfection and purified for the LC-MS analysis.Table 10: DNA chain mixes used for antibody production via transient transfection in Example 4. The plasmid mixes resulting in the individual samples are depicted below with their corresponding chain mass ratio used for transient co-transfection, e.g. the wildtype antibody IgG mix was assembled from expression of the plasmids including the sequences DNA-1 , DNA-22, DNA-39 and DNA-40. The row with the sample names specifies which model antibody harbors the CH1 and CL domain mutations.4.2 Purification of Model Antibody IgG Constructs

[0242] In preparation for the LC-MS analysis the samples produced in 4.1 were purified using a Protein A HP SpinTrap Column (Cytiva) with a 20 mM NaH2PO4 pH 7.2 binding buffer, a 20 mM sodium citrate pH 2.9 buffer and a 1 M Tris pH 9 buffer. All centrifugation steps were 30 s at 100 x g. First, the storage solution was removed via centrifugation. Then, the columns were equilibrated with600 pL binding buffer via centrifugation. Sample was bound to the column by pipetting 600 pL of sample onto the column, incubation for 4 min at room temperature, centrifugation and collection of the flowthrough. This was repeated until 3 mL of sample had been applied to the column. The column was then washed twice with 600 pL binding buffer. Antibodies were eluted using 200 pL of elution buffer and 15 pL of neutralization buffer to achieve a pH between 7 and 8. The first two elution fractions were pooled and subsequently analyzed for concentration and purity via Nanodrop UV-spectrophotometry (Thermo) and SDS-PAGE.4.3 LC-MS measurement of Model Antibody IgG Constructs

[0243] All purified samples were diluted to 0.2 mg / mL by addition of 10 mM Tris buffer at pH 7.5. Additionally, 1.5 pL of a 200 mM calcium chloride solution was added to all samples to a final concentration of 10 mM. FabDELLO enzyme (Genovis) was added with either the recommended enzyme concentration, according to the manufacturers protocol, or up to 10 % excess and the samples were incubated for 2 h at 37 °C. Per sample 0.4 pg of digested protein was applied to a 1 x 100 mm Acquity UPLC Protein BEH C4 column (Waters) heated to 80°C and separated by RP-HPLC on an Acquity l-Class UPLC System (Waters). A linear gradient of acetonitrile from 27 to 33 % was run over7.3 min at a flow rate of 0.15 ml / min, 0.1 % (v / v) difluoroacetic acid was used as a modifier. Eluting proteins were analyzed on a Xevo G2-XS QTOF mass spectrometer (Waters) operated in positive ion, sensitivity mode. Full scan mass spectra were acquired from 750 to 3000 m / z with Glu-Fibrinopeptide peptide B as a lock mass. The resulting mass spectrometry data were deconvoluted and analyzed in ProteinMetrics Inc. Byos intact module with the assumption that all possible disulfide bridges had formed. The masses of the four different possible antibody species in each mix were calculated using the Geneious Prime software (Graph Pad Software LLC) and attributed to the different mass peaks in the LC-MS analysis to calculate the correct pairing from the relative peak signals.4.4 Results

[0244] Analysis of the LC-MS peak signal data shows a proportion of 62 % of correctly paired antibody species if the two model antibody wildtype variants are co-expressed (Figure 10), while the mispairing variants MP1 , heavy chain of model antibody 1 and light chain of model antibody 3, and MP2, heavy chain of model antibody 3 and light chain of model antibody 1 , could clearly be matched individually in the mass analysis, showing the feasibility of detecting all four possible antibody species. The model antibody mix with the charge variant mutations (CV) shows an enhanced correct pairing compared to the wildtype of up to 81 %. The samples with the leads chosen for the LC-MS analysis, Lead 1 , Lead2 and Lead3 all show an enhancement of the ratio of correct pairing of CH1 and CL domains compared to the wildtype.Example 5: Assessment of retained antigen binding capacity after introduction of CH1-CL mispairinq prevention mutations via FACS-binding assay

[0245] The present example shows the results of the antigen binding analysis of selected model antibodies containing the Lead 1 -6 mutational pair CH 1 and CL mutations in comparison to the wildtype model antibodies. For this purpose, plasmids encoding the CH1 and CL sequence with the mispairing prevention mutations as cloned within Example 3, were produced individually, via transient cotransfection. The concentrations of assembled model Ab1 and model Ab3 species secreted into the cell culture supernatant were determined by making use of a sandwich immunoassay (GyroLab ELISA) using anti-Fc detection antibodies. Afterwards, the antigen binding capacities of the different model Ab1 and model Ab3 species were analyzed in a flow cytometric binding assay.

[0246] Methods of the present example include:5.1 Production of Model Antibody IgG Constructs via transient Transfection

[0247] Expression of IgG model Ab1 and model Ab3 antibodies with wildtype CH1-CL sequences or Lead1-Lead6 mutations' sequences was performed using the plasmid DNAs described in Example3.1 (Table 7) via transient transfection with plasmid mixes encoding for the respective IgG model antibody heavy and light chain in three biological replicates, as shown in Table 11 , according to the manufacturers manual for the 293ExpiF expression system according to the previously described methods (see Example 1.2 and 2.2). Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers. Cell culture supernatant containing the differently assembled antibody species was collected 4 or 5 days after transfection and directly used for further Gyros ELISA analysis.Table 11 : DNA chain mixes used for antibody production via transient transfection in Example 5. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of HEK cells for the expression of wildtype IgG chains of model Ab1 or model Ab3 as well as for Lead 1- Lead6 mutations-containing IgG chains.5.2 Gyros ELISA for Model Antibody IgG Constructs Quantitation

[0248] The concentrations of correctly paired model Ab1 and model Ab3 antibodies were analyzed using the Gyros xPand™ XPA1055 immunoassay system (GyroLab) with a Bioaffy CD20HC disc (GyroLab) and the huIgG Low or High Titer Kit (GyroLab). Total antibody concentration was determined using biotinylated anti-human IgG-Fc antibody (Thermo Fisher Scientific) as the capture reagent and an AlexaFluor647 coupled anti-human IgG-Fcy (Jackson ImmunoResearch Europe Ltd) as the detection reagent. Standard curves for total antibody concentration were measured with previously produced and purified model Ab1 and model Ab3 separately. 5.3 FACS-based cellular binding assay for assessment of antigen binding capacity of modelAb1 and model Ab3 with wildtype sequence and Lead1-Lead6 mutations

[0249] The binding of model Ab1 and model Ab3 antibodies to target-expressing cells was assessed by flow cytometric analysis. The cells were thawed and cultured in assay medium consisting of RPMI Glutamax supplemented with 10% heat-inactivated FBS (both from Gibco) for at least two passages. On the day of the experiment, the cells were harvested, washed, and transferred into a 96-well plate (5 x 105cells / well). The cells were then incubated with the test items as primary antibodies at ten different dilutions for 30 min at 2-8°C. After incubation, the cells were washed twice with FACS buffer, resuspended in 100 pL FACS buffer and incubated with an APC-conjugated goat anti-human IgG (Jackson ImmunoResearch Europe Ltd) antibody (1 :200 dilution) for 30 min at 2-8°C in the dark. Dead cells were stained by adding eFluor 506 (1 :400 dilution) to the reaction in parallel with the secondary antibody. The cells were then washed twice with PBS and fixated by adding 100 pL of BD fixative solution. The cells were then analyzed by flow cytometry using a BD FACSCelesta (BD Biosciences). Cells were first gated for singlets followed by gating viable cells and APC-positive cells.5.4 Results

[0250] Target-specific cell binding of model Ab1 and model Ab3 antibodies was determined by flow cytometric binding assays, model Ab1 or model Ab3 antibody-containing cell culture supernatant was serially diluted and incubated with target-expressing cells. The binding curves of model Ab1 and model Ab3 antibodies containing wildtype CH1-CL sequences were then measured and overlayed with the curves of model Ab1 and model Ab3 antibodies containing Lead1-6 mutations' sequences.

[0251] All model Ab1 and model Ab3 antibodies showed dose-dependent binding to target-expressing cells (Fig. 11-16).

[0252] The maximal signals and EC50 values are essentially unchanged or below factor 2, respectively, between any of the model Ab1 and model Ab3 antibodies containing Lead1-6 mutations' sequences in comparison to the respective model antibodies containing wildtype CH1-CL sequences.

[0253] As the binding curves of WT and Lead1-Lead6 model Ab1 and model Ab3 antibodies overlap, it can be assumed that introduction of any of the Lead1-6 mutations into the CH1-CL domains has no impact on the antigen-specific binding ability of the Fab domain and thus the antibody functionality is retained.Example 6: Screening of additional CH1-CL mutation pairs for antibody productivity and CH1-CL mispairinq prevention properties via Western Blot-based method

[0254] The present example shows the results of the screening of additional candidate mutation pairs, named Lead 7-16, for the prevention of CH 1 -CL mispairing. For the comparison of the effects of the different mutations on antibody mispairing, the established Western Blot-based assay described in Example 1 has been utilized. To avoid heavy chain to heavy chain mispairing the assay is conducted using a wildtype IgG model Ab1 and a second model Ab2 expressed as a Fab as also described in Example 1 and 2. The different CH1 and CL mutations were introduced via cloning in the IgG model Ab1 CH1 and CL sequences, resulting in the plasmid constructs according to Table 12. With theplasmid ratios described in Table 13, the IgG / Fab mixes were expressed in parallel in HEK 293ExpiF cells via transient transfection. The resulting supernatants were analyzed for antibody titers via automated ELISA to assess differences in expression compared to the wildtype Fab or literature controls. To compare mispairing between the candidates, equal amounts of each co-transfection mix were analyzed on non-reduced Western Blots and the band signal intensities were determined via image analysis as described in Example 2. The ratio of the value of the additional bands with mispairing species and that of all detected bands, except those species that missed light chains, was calculated to obtain the mispairing proportions.

[0255] Methods of the present example include: 6.1 Cloning of DNA constructs

[0256] Cloning was performed into the mammalian expression vector pcDNA3.4-TOPO by techniques that are commonly known by people skilled in the art. Most constructs were cloned according to the in vivo assembly method explained in Example 1.1 and 2.1 , while the remaining constructs, especially the controls, were cloned using overhang PCR and restriction cloning. The resulting constructs are displayed in Table 12.Table 12: Overview of cloned DNA constructs for antibody production used for screening ofCH1-CL domain Lead7-16 mutation pairs.6.2 Production of Model Antibody IgG and Fab Construct Mixes via transient Transfection

[0257] Production of IgG model Ab1 and Fab model Ab2 antibodies with CH1 -CL sequences either of wildtype, control, lead or candidate sequences was performed using plasmid mixes encoding for the respective IgG model antibody heavy and light chain and the wildtype Fab chains of model Ab2 via transient transfection, as shown in Table 13, according to the manufacturers manual for the 293ExpiF expression system and as described previously (see Example 1 .2 and 2.2). Samples were produced at a 500 pL scale in a 96-well 2 mL deep well plate format using a customized automated liquid handler Biomek i7 (Beckman Coulter) in three biological replicates. Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers. Cell culture supernatant containing the differently assembled antibody species was collected four or five days of cultivation at 37 °C and 5% CO2 after transfection and directly used for further automated sandwich ELISA and Western Blot analysis.Table 13: DNA chain mixes used for antibody production via transient transfection in Example 6. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of HEK cells for the expression of wildtype IgG chains of model Ab1 and Fab chains of model Ab2 as well as for antibody mixes containing CH1-CL domains with either the X or CV reference mutations or Lead4 or Lead7-16 mutations.6.3 Western Blot analysis of CH1-CL mispairing

[0258] The comparative analysis of the mispairing of the CH1 and CL domains in the samples illustrated in Example 6.2. was conducted according to the method described in Example 2.4. The results of the Western Blot analysis were verified in a second experiment.6.4 Results of antibody expression and Western Blot mispairing assay

[0259] Expression analysis of the different controls and samples compared to the mix of the wildtype chains of model Ab1 and model Ab2 shows similar titers for most leads (Figure 17). Only LeadlO and Leadl 1 showed lower titers than the wildtype, with 77.5 % and 44.7 % respectively, a decrease in titer comparable to that seen with the DM mutations. The different samples comprised of two chains of themodel Ab1 IgG with mutations in the CH1 and CL domain and the two chains of the model Ab2 Fab, equal total antibody masses were applied of each sample. Candidate samples were analyzed on two different Western Blots (Figure 18 and 19) together with the wildtype IgG and Fab mix (WT), the CrossMab charge variant (CV), and Lead4 as controls on both blots, while the domain swap variant (X) and the DuetMab variant (DM) were only applied to one of the blots. DM, CV and X were used as reference controls to compare novel mispairing prevention candidates to common technologies used in the literature (WO2015150447A1 , WO2017055539A1 and WO2013096291 A2 ) The wildtype sample (WT) shows the expected statistical mispairing of around 30 to 40 % for the two model antibodies, with mostly the mispaired species present, that comprises of model Ab1 heavy chain and the model Ab2 light chain. All samples and controls show both the correctly paired IgG band at around 150 kDa and the correctly paired Fab band at around 65 kDa. Two mispairing bands greater in size than the IgG band, due to the presence of the heavier light chain of the Fab, could be detected in some of the samples and the mispaired species with the Fab heavy chain and the smaller IgG light chain could be seen at around 40 kDa. The control sample band patterns of the WT, the CV and the Lead4 control are similar on all analyzed blots and show a similar ratio of correct pairing when the Western Blot signals were analyzed according to the method description, demonstrating the reproducibility of the assay. After two experiments, the ten candidates with the lowest ratio of mispaired species were selected for further analyses. Especially the candidates Lead14-16 performed best, with almost no mispaired species detectable (Table 14). All selected leads showed a small amount of mispairing species of around 2 to 14 %, even with longer detection times. Generally, the Western Blot mispairing assay tends to overestimate the ratio of mispaired species, but has advantages for comparison of the candidates, due to its sensitivity for mispaired species’ bands.Table 14: Overview of selected lead candidates and their CH1 and CL domain mutations with correct pairing proportions based on additional results from Example 6.Example 7: Assessment of antibody domain mispairinq proportions of additional novel sets of CH1- CL mutation pairs via ELISA-based method

[0260] The present example shows the results of the assessment of antibody domain mispairing proportions after co-transfection of a mixture of four DNA plasmids encoding two different full IgG model antibodies with either wildtype or additional mutated CH1 and CL domains. For this purpose, plasmids encoding the CH1 and CL sequence with the additional mispairing prevention mutations, as generated in previous examples were produced in mixes of wt-HC1 +wt-LC1+neo-HC2+neo-LC2 or wt-HC2+wt-LC2+neo-HC1+neo-LC1 via transient co-transfection along with control mixes with the plasmid ratios described in Table 15. IgG chains and assembled model Ab1 and model Ab3 species secreted into the cell culture supernatant were analyzed by making use of the sandwich immunoassay described in Example 3. The proportion of correctly paired Fab domains was calculated by dividing the sum of both individual antibody concentrations measured via anti-idiotype antibodies by the total antibody concentration.

[0261] Methods of the present example include:7.1 Production of Model Antibody IgG Constructs via transient Transfection

[0262] Production of IgG model Ab1 and IgG model Ab3 antibodies with CH 1 -CL sequences either of wildtype, control, lead or candidate mutation sequences was performed using plasmid mixes encoding for the respective IgG model antibody heavy and light chain and the wildtype IgG chains of model Ab3 via transient transfection in three biological replicates, as shown in Table 15, according to the manufacturers manual for the 293ExpiF expression system according to the previously described methods (see Example 1.2 and 2.2). Samples were produced at a 500 pL scale in a 96-well 2 mL deep well plate format using a customized automated liquid handler Biomek i7 (Beckman Coulter). Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers. Cell culture supernatant containing the differently assembled antibody species was collected four or five days of cultivation at 37 °C and 5% CO2 after transfection and directly used for further Gyros ELISA and Western Blot analysis.Table 15: DNA chain mixes used for antibody production via transient transfection in Example 7. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of HEK cells for the expression of wildtype IgG chains of model Ab1 and Fab chains of model Ab2 as well as for antibody mixes containing CH1-CL domains with either the X or CV reference mutations or Lead4 or Lead7-16 mutations.7.2 anti-ID ELISA measurement of Model Antibody IgG Constructs

[0263] The analysis of CH1 and CL domain mispairing in the antibody mix produced as described inExample 7.1 was analyzed according to the method described in Example 3.3 7.3 Results

[0264] The properties of the different candidate CH1 and CL domain mutations were evaluated in an ELISA mispairing assay and compared to the wildtype, the literature controls, DuetMab technology (DM), the CrossMab charge variant mutations (CV), the domain swap variant (X), and Lead4 from the first set of lead mutations. At least six replicates were analyzed for each of the samples. For the controls nine replicates and for the wildtype and the Lead4 mutations 15 replicates were analyzed. Asexpected the mispairing controls MP1 and MP2 showed no correct pairing, meaning no background binding of the anti-idiotype antibody to the mispaired species, while not affecting the determination of the total antibody concentration. The wildtype shows a correct pairing of 67.6 % (Figure 20 and 21), which is in line with the previous results in all assays, which show correct pairing of around 60 to 70 % for the wildtype mix of model Ab1 and Ab3. All control samples show enhanced correct pairing compared to the wildtype CH 1 and CL sequences, with the DM sample showing 78.0 % correct pairing, the domain swap variant (X) 92.0 % and the charge variant mutations (CV) sample even 101.4 %. Values of over a 100 % could be the results of variations of error between the different ELISA measurements, e.g. of the total antibody concentration underestimated and the individual Fab detection with anti-idiotype antibodies overestimated. This is relativized by employing the standard curve with exact prepared mispairing mixes measured in parallel with every ELISA, but can nevertheless, lead to values higher than 100 %. Lead4 shows a similar score as the domain swap variant (X), with 91 .1 % correct pairing. Many of the candidates and those that were ultimately chosen as leads show the capability to ensure correct pairing of over 90 %, with the best candidate Leadl 3 even showing 96.4 % correct pairing. Many of the candidates show lower correct pairing than the control sequences, with some even performing worse than seen with the wildtype sequences. This worse performance could be attributed to the mutations either favouring the formation of mispaired species over those of correct species or causing deviations in ELISA analysis due to impacts on the VH and VL domain formation and thus impaired binding of the anti-idiotype antibody.Example 8: Assessment of Fab domain pairing after introduction of selected additional sets of CH1- CL mispairinq prevention mutations via LC-MS analysis

[0265] The present example shows the results of the CH1 and CL domain mispairing detected via LC-MS mispairing of a mix of two antibodies, one with the wildtype CH1 and CL domain sequences, the other with the mutations of Lead4-16, reference control sequences or another wildtype. The model antibodies used were either a mix of IgG model Ab1 and IgG model Ab3 or Fab model Ab1 and Fab model Ab2. The samples were generated via transient transfection of HEK cells. In preparation for the LC-MS analysis, the sample supernatants were purified via affinity chromatography, the antibody concentration determined via UV-spectrophotometry and the Fc domains, if present, cut by means of enzyme digestion. Equal amounts of each antibody mix were loaded onto an RP-HPLC column and analyzed in a QTOF mass spectrometer. The LC-MS mass peak signal comparison of the known individual antibody species’ masses allowed for the determination of heavy and light chain or CH 1 -CL mispairing.

[0266] Methods of the present example include:8.1 Cloning of DNA constructs

[0267] Cloning was performed by techniques that are commonly known by people skilled in the art into the mammalian expression vector pcDNA3.4-TOPO. The constructs were cloned using aspreviously described (Example 1 and 2), but the insert was generated via overhang PCR and assembled with the digested vector via enzymatic ligation, according to standard procedures well known by the person skilled in the art. The resulting constructs are displayed in Table 16.Table 16: Overview of cloned DNA constructs for antibody production used for screening of additional CH1-CL domain mutation pairs in the Fab format via LC-MS analysis.8.2 Production of Model Antibody IgG or Fab Constructs via transient Transfection

[0268] Mispairing evaluation was conducted by mixing heavy and light chains of the full IgG model Ab1 and model Ab3 antibodies. For each CH1-CL mispairing prevention variant, the corresponding mutations were introduced to model Ab1 antibodies, while the other model Ab3 antibody had the wildtype CH1 and CL sequences of a human kappa lgG1 antibody. Constructs with mutations near the hinge region had to be processed differently, since the digestion of the hinge region by the FabDello enzyme (Genovis) in preparation of the LC-MS analysis was hindered by such mutations. For these samples, mixes of the heavy and light chains of the model Ab1 Fab containing CH1-CL mispairing preventionmutations and the heavy and light chains of the wildtype model Ab2 Fab were produced with the same method as the IgG mixes.Expression of IgG model Ab1 and model Ab3, or respectively Fab model Ab1 and Fab model Ab2, chain mixes was performed via transient transfection of HEK cells with a plasmid mix encoding for the four model antibody chains, as shown in Table 17, according to the manufacturers manual for the 293ExpiF expression system (Thermo Scientific) according to the previously described methods (see Example 1 .2 and 2.2), albeit only one 5 mL replicate each in 50 mL tube bioreactors with membrane caps (TPP) was produced. Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers from the different plasmid backbones to prevent biases in the assay from missing or underrepresented chains. Samples that showed very low amount of a certain chain were not considered. Cell culture supernatants containing the differently assembled antibody species were collected 4 or 5 days after transfection and purified for the LC-MS analysis.Table 17: DNA chain mixes for antibody production via transient transfection in Example 8. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of HEK cells HEK cells for the expression of IgG or Fab chains containing either wildtype or control or Lead7- Lead16 mutations.8.3 Purification of Model Antibody IgG Constructs

[0269] The culture supernatants with the antibody mixes described in Example 8.2 were prepared for LC-MS analysis using purification via affinity chromatography. The mixes of the lgG1 variants of the model antibodyl and 3 were purified according to the method described in Example 4.2.

[0270] The mixes of Fab model antibodies Ab1 and Ab2 were purified via affinity chromatography using CH1-XL columns (Thermo Fisher Scientific). The flow rate was 0.5 mL min-1during loading and 1 mL min-1during the other steps. Equilibration was conducted with 4 column volumes (cv) of 20 mm Tris buffer pH 7, the column was washed with 4 cv of 20 mM Tris buffer pH 8. Samples were eluted via pH shift with a 20 mM acetic acid buffer pH3.5 with 150 mM NaCI. Each 1 mL elution fraction was neutralized with 60 pL of 1 M Tris buffer pH 9. After each purification method the samples were analyzed via SDS-PAGE and the concentration determined via Nanodrop UV-spectrophotometry (Thermo).8.4 LC-MS measurement of Model Antibody IgG Constructs

[0271] All purified samples were diluted to 0.2 mg / mL by addition of 10 mM Tris at pH 7.5. The CH1 and CL domain mispairing of the samples were determined in a mass spectrometer according to the method described in Example 4.3.

[0272] In the case of the Fab model antibody mixes, the digestion with FabDello was not necessary.8.5 Results

[0273] The samples comprising of IgG mixes and the samples with Fab mixes were measured separately. The analysis of the LC-MS peak signal data of the wildtype model antibody 1 and 3 IgG mixes showed a proportion of 61 % of correctly paired antibody species if the two model antibody wildtype variants were co-expressed (Figure 22). The purified mispairing variant mixes with only HC1 and LC2 (MP1) and HC2 and LC1 (MP2) showed only the single mass peak corresponding to each protein species. All evaluated samples could convey an increase of correctly paired CH1 and CL domains, with Lead4 and Lead8 at around 93 %, Lead7 at 95 %, Lead9 and Lead12 at 97%. For the rest of the samples no mispaired species could be detected. The analysis of the Fab model antibody 1 and 2 mixes were analyzed in the same way as the IgG mixes (Figure 23) The sample of Lead4 with the mixes of the Fab model antibody 1 and 2 showed 96% of correct pairing (cf. 93% with IgG antibody 1 and 3), thus performing better than the Duetmab control (DM) sample with 91 %. In either of the samples of Lead5, Lead6, Lead14, Lead15 and Lead16 signals corresponding to mispaired species could not be detected.Example 9: Assessment of retained antigen binding capacity after introduction of Lead 7-16 CH1-CL mispairing prevention mutations via FACS-binding assay

[0274] The present example shows the results of the antigen binding analysis of selected model antibodies containing the Lead7-16 mutational pair CH1 and CL mutations in comparison to the wildtype model antibodies. For this purpose, plasmids encoding the CH1 and CL sequence with themispairing prevention mutations as cloned within Example 7, were produced individually via transient co-transfection. The concentrations of assembled model Ab1 species secreted into the cell culture supernatant were determined by making use of a sandwich immunoassay (GyroLab ELISA) using anti-Fc detection antibodies. Afterwards, the antigen binding capacities of the different model Ab1 species were analyzed in a flow cytometric binding assay.

[0275] Methods of the present example include:9.1 Production of Model Antibody IgG Constructs via transient Transfection

[0276] Expression of IgG model Ab1 antibodies with wildtype CH1 -CL sequences or Lead7-Lead16 mutations' sequences was performed using the plasmid DNAs described in Example 6 (Table 12) via transient transfection with plasmid mixes encoding for the respective IgG model antibody heavy and light chains, as shown in Table 18, according to the manufacturers manual for the 293ExpiF expression system according to the previously described methods (see Examples 1.2 and 2.2). Plasmid titrations were conducted to make sure the individual antibody chains were expressed with comparable titers. Cell culture supernatant containing the differently assembled antibody species was collected four or five days after transfection and directly used for further Gyros ELISA analysis.Table 18: DNA chain mixes used for antibody production via transient transfection in Example 9. Plasmid mixes and their respective chain mass ratio used for transient co-transfection of HEK cells for the expression of wildtype IgG chains as well as for Lead7-Lead16 mutations-containing IgG chains of model Ab1 .9.2 Gyros ELISA for Model Antibody IgG Constructs Quantitation

[0277] The concentrations of model Ab1 antibodies were analyzed using the Gyros xPand™ XPA1055 immunoassay system (GyroLab) with a Bioaffy CD20HC disc (GyroLab) and the huIgG Low or High Titer Kit (GyroLab) as described in Example 5.9.3 FACS-based cellular binding assay for assessment of antigen binding capacity of modelAb1 with wildtype sequence and Lead7-Lead16 mutations

[0278] The binding of model Ab1 antibodies to target expressing cells was assessed by flow cytometric analysis as described in Example 5.9.4 Results

[0279] Target-specific cell binding of model Ab1 antibody species with either wildtype CH1 and CL domains or Lead7-16 mutations-containing CH1 and CL domains was determined by flow cytometric binding assays, model Ab1 antibody-containing cell culture supernatant was serially diluted and incubated with target-expressing cells. The binding curves of model Ab1 antibodies containing wildtype CH1-CL sequences were then measured and overlayed with the curves of model Ab1 antibodies containing Lead7-16 mutations' sequences.

[0280] All different model Ab1 antibody samples showed dose-dependent binding to targetexpressing cells (Fig. 24 and 25).

[0281] The maximal signals and EC50 values are essentially unchanged or below factor 2, respectively, between any of the model Ab1 containing Lead7-16 mutations' sequences in comparison to the respective model Ab1 containing wildtype CH1-CL sequences.

[0282] As the binding curves of WT and Lead7-16 model Ab1 antibodies overlap, it can be assumed that introduction of any of the Lead7-16 mutations into the CH1-CL domains has no impact on the antigen-specific binding ability of the Fab domain and thus the antibody functionality is retained.

Claims

CLAIMS1. A pharmaceutical composition comprising (a) at least one nucleic acid encoding at least a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain or a particle comprising said at least one nucleic acid, and (b) optionally at least one pharmaceutically acceptable excipient, wherein the at least one nucleic acid encodes an IgG antibody or an IgG antibody fragment thereof, wherein the IgG antibody or IgG antibody fragment thereof comprises the CH1 domain and the CL domain, or a fusion protein comprising said IgG antibody or IgG antibody fragment thereof, wherein the CH1 domain and the CL domain are selected from the group consisting of(1)(i) a CH1 domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (1)(ii) a CL domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU; preferably (1a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU, and (1 a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (1 b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU, and (1 b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU;(2)(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU, and (2)(ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU;(3)(i) a CH1 domain comprising the amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU, and (3)(ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU;(4)(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU, and (4)(ii) a CL domain comprising the amino acidsubstitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU;(5)(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU, and (5)(ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU;(6)(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU, and (6)(ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU;(7)(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU, and (7)(ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU;(8)(i) a CH1 domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F 170C and C220V in the numbering according to EU, and (8)(ii) a CL domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU; preferably (8a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU, and (8a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU;(9)(i) a CH1 domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185W in the numbering according to EU or (i”) G141 Wand K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’") A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S131 I and V185W in the numbering according to EU, and (9)(ii) a CL domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU; and(10)(i) a CH1 domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (10)(ii) a CL domain comprising the amino acid substitutions E123K, N137L, T164Mand T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU.

2. The pharmaceutical composition according to claim 1 , wherein the at least one nucleic acid encodes an lgG1 antibody or lgG1 antibody fragment thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the CL domain is of kappa isotype.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the at least one nucleic acid encodes an antibody selected from the group consisting of a symmetric antibody and an asymmetric antibody.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the at least one nucleic acid encodes an antibody fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof.

7. The pharmaceutical composition according to any one of claims 1 to 3 or 6, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric antibody-peptide-i-8 (preferably symmetric antibody-peptide2), asymmetric antibody- peptide-i-8 (preferably asymmetric antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1- CL), CH1-CL-peptidei-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2) .

8. The pharmaceutical composition according to claim 6 or 7, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

9. The pharmaceutical composition according to any one of claims 1 to 3 or 6 to 8, wherein the fusion protein is selected from the group consisting of symmetric antibody-cytokine2, symmetric antibody-scFv2, symmetric antibody-VHH2, asymmetric antibody-cytokine2, asymmetric antibody-scFv2, asymmetric antibody-VHH2, Fab-cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2-VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1-CL-VHH, scFv-CH1-CL-scFv, VHH- CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2-CH1-CL-scFv2, VHH2-CH1-CL- VH H2, SCFV2-CH 1 -CL-VHH2 and VH H2-CH 1 -CL-scFv2.

10. The pharmaceutical composition according to any one of claims 1-3, wherein the at least one nucleic acid encodes an antibody or fragment thereof, or a fusion protein comprising said antibody or fragment thereof, wherein the antibody or fragment thereof comprises at least two different CH1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are selected from the group consisting of (1)(i) and (1 )(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each additional CH1 domain comprises a CH1 domain different from (1)(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and each additional CL domain comprises a CL domain different from (1)(ii), (2)(ii), (3)(ii), (4)(ii), (5)(ii), (6)(ii), (7)(ii), (8)(ii), (9)(ii), and (10)(ii).

11. The pharmaceutical composition according to claim 10, wherein each additional CH 1 domain matches an additional CL domain.

12. The pharmaceutical composition according to claim 10 or 11 , wherein an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein the encoded antibody or fragment thereof is selected from the group consisting of an asymmetric antibody and a F(ab’)2 fragment.

14. The pharmaceutical composition according to any one of claims 10 to 12, wherein the at least one nucleic acid encodes a fusion protein, wherein a peptide is fused to the N- and / or C- terminus of the antibody or fragment thereof.

15. The pharmaceutical composition according to claim 10 or 14, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) asymmetric antibody-peptide and F(ab’)2-peptide.

16. The pharmaceutical composition according to claim 14 or 15, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

17. The pharmaceutical composition according to any one of claims 10 and 14 to 16, wherein the fusion protein is selected from the group consisting of asymmetric antibody-cytokine2, asymmetric antibody-scFv2, asymmetric antibody-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, and F(ab’)2-VHH2.

18. The pharmaceutical composition according to any one of claims 10 to 17, wherein the encoded antibody or fragment thereof or fusion protein comprises two different CH1 domains and two different CL domains.

19. The pharmaceutical composition according to any one of claims 1 to 9, wherein the at least one nucleic acid encodes at least two antibodies or fragments thereof, or fusion proteins comprising the antibody or fragment thereof, wherein each CH1 domain and each CL domain of an encoded first antibody or fragment thereof or fusion protein is selected from the group consisting of (1)(i) and (1)(ii), (2)(i) and (2)(ii), (3)(i) and (3)(ii), (4)(i) and (4)(ii), (5)(i) and (5)(ii), (6)(i) and (6)(ii), (7)(i) and (7)(ii), (8)(i) and (8)(ii), (9)(i) and (9)(ii), and (10)(i) and (10)(ii); and wherein each CH1 domain and each CL domain of each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain different from ((1)(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i), and a CL domain different from (1 )(i), (2)(i), (3)(i), (4)(i), (5)(i), (6)(i), (7)(i), (8)(i), (9)(i), and (10)(i).

20. The pharmaceutical composition according to claim 19, wherein each additionally encoded antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additionally encoded antibody or fragment thereof or fusion protein.

21. The pharmaceutical composition according to claim 19 or 20, wherein an additionally encoded antibody or fragment thereof or fusion protein comprises a wild-type CH1 domain and a wildtype CL domain.

22. The pharmaceutical composition according to any one of claims 19 to 21 , wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an IgG antibody or IgG antibody fragment thereof.

23. The pharmaceutical composition according to any one of claims 19 to 22, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an IgG 1 antibody or lgG1 antibody fragment thereof.

24. The pharmaceutical composition according to any one of claims 19 to 23, wherein the CL domain of the at least one additionally encoded antibody or fragment thereof or fusion protein is of kappa isotype.

25. The pharmaceutical composition according to any one of claims 19 to 24, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is an antibody selected from the group consisting of a symmetric IgG antibody and an asymmetric IgG antibody.

26. The pharmaceutical composition according to any one of claims 19 to 24, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.

27. The pharmaceutical composition according to any one of claims 19 to 24, wherein the at least one additionally encoded antibody or fragment thereof or fusion protein is a fusion protein,wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof.

28. The pharmaceutical composition according to claim 27, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric IgG antibody- peptide-i-8 (preferably symmetric IgG antibody-peptide2), asymmetric IgG antibody-peptide-i-8 (preferably asymmetric IgG antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1- CL), CH1-CL-peptidei-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2-CH1-CL-peptide2) .

29. The pharmaceutical composition according to claim 27 or 28, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

30. The pharmaceutical composition according to any one of claims 19 to 24 and 27 to 29, wherein the fusion protein is selected from the group consisting of symmetric IgG antibody-cytokine2, symmetric IgG antibody-scFv2, symmetric IgG antibody-VHH2, asymmetric IgG antibody- cytokine2, asymmetric IgG antibody-scFv2, asymmetric IgG antibody-VHH2, Fab-cytokine, Fab- scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, F(ab’)2-VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1-CL-cytokine, CH1-CL-scFv, CH1-CL- VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv-CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2- CH 1 -CL-SCFV2, VH H2-CH 1 -CL-VHH2, scFv2-CH 1 -CL-VH H2and VH H2-CH 1 -CL-scFv2.31 . The pharmaceutical composition according to any one of claims 1 to 30, wherein at least a CH1 domain is encoded by a first nucleic acid and at least a CL domain is encoded by a second nucleic acid.

32. The pharmaceutical composition according to any one of claims 1 to 31 , wherein the nucleic acid is DNA or RNA, preferably mRNA.

33. The pharmaceutical composition according to any one of claims 1 to 32, wherein the particle is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a baculoviral vector, an Epstein-Barr viral vector, a poxvirus vector, a virosome, a lipid nanoparticle (LNP), a liposome, a lipoplex (LPX), and a polyplex (LPX).

34. The pharmaceutical composition according to any one of claims 1 to 33 for use in therapy.

35. The pharmaceutical composition according to any one of claims 1 to 33 for use in the treatment of cancer.

36. The pharmaceutical composition according to any one of claims 1 to 33 for use in the treatmentof an infectious disease.

37. The pharmaceutical composition according to any one of claims 1 to 33 for use in the treatment of an autoimmune disease.

38. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU; preferably (a)(i) a CH1 domain comprising the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in the numbering according to EU, and (a)(ii) a CL domain comprising the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU; or (b)(i) a CH1 domain comprising the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU, and (b)(ii) a CL domain comprising the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU.

39. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU.

40. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions S130M, G141W and H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU.41 . An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutionsF118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU.

42. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions (i’) L124I, G141 W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU.

43. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU.

44. An antibody or a fragment thereof comprising(i) a CH1 domain comprising the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU, and (ii) a CL domain comprising the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU.

45. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU; preferably (a)(i) a CH1 domain comprising the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU, and (a)(ii) a CL domain comprising the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU.

46. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T139W, T169V and V185Win the numbering according to EU or (i”) G141 Wand K222R in the numbering according to Kabat corresponding to G143Wand K214R in the numbering according to EU or (i’”) A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S1311 and V185W in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU.

47. An antibody or a fragment thereof comprising(i) a constant heavy chain 1 (CH1) domain comprising the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU, and (ii) a constant light chain (CL) domain comprising the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU.

48. The antibody or fragment thereof according to any one of claims 38 to 47, wherein the antibody or fragment thereof is an IgG 1 antibody or IgG 1 antibody fragment thereof.

49. The antibody or fragment thereof according to any one of claims 38 to 48, wherein the CL domain is of kappa isotype.

50. The antibody or fragment thereof according to any one of claims 38 to 49, wherein the antibody or fragment thereof is an antibody selected from the group consisting of a symmetric antibody and an asymmetric antibody.51 . The antibody or fragment thereof according to any one of claims 38 to 49, wherein the antibody or fragment thereof is a fragment selected from the group consisting of a Fab fragment, a F(ab’)2 fragment and a CH1-CL fragment.

52. A fusion protein comprising the antibody or fragment thereof according to any one of claims 38 to 51.

53. The fusion protein according to claim 52, wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof.

54. The fusion protein according to claim 52 or 53, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) symmetric antibody-peptide-i-8 (preferably symmetric antibody-peptide2), asymmetric antibody-peptide-i-8 (preferably asymmetric antibody-peptide2), Fab-peptide-M (preferably Fab-peptide2), F(ab’)2-peptidei-8 (preferably F(ab’)2-peptide2), peptidei-2-CH1-CL (preferably peptide2-CH1-CL), CH1-CL-peptide-i-2 (preferably CH1-CL-peptide2) and peptidei-2-CH1-CL-peptidei-2 (preferably peptide2- CH1-CL-peptide2) .

55. The fusion protein according to claim 53 or 54, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

56. The fusion protein according to any one of claims 52 to 55, wherein the fusion protein is selected from the group consisting of symmetric antibody-cytokine2, symmetric antibody-scFv2, symmetric antibody-VHH2, asymmetric antibody-cytokine2, asymmetric antibody-scFv2, asymmetric antibody-VHH2, Fab-cytokine, Fab-scFv, Fab-scFv2, Fab-VHH, Fab-VHH2, F(ab’)2- cytokine2, F(ab’)2-scFv2, F(ab’)2-VHH2, cytokine-CH1-CL, scFv-CH1-CL, VHH-CH1-CL, CH1- CL-cytokine, CH1-CL-scFv, CH1-CL-VHH, scFv-CH1-CL-scFv, VHH-CH1-CL-VHH, scFv- CH1-CL-VHH, VHH-CH1-CL-scFv, scFv2-CH1-CL-scFv2, VHH2-CH1-CL-VHH2, scFv2-CH1- CL-VHH2and VHH2-CH1-CL-scFv2.

57. The antibody or fragment thereof according to any one of claims 38-49 wherein the antibody or fragment thereof comprises at least two different CH1 domains and at least two different CL domains, wherein a first CH1 domain and a first CL domain are (i) and (ii), and wherein each additional CH1 domain comprises a CH1 domain different from (i) and each additional CL domain comprises a CL domain different from (ii), wherein the antibody or fragment thereof preferably comprises two different CH1 domains and two different CL domains.

58. The antibody or fragment thereof according to claim 57, wherein each additional CH1 domain matches an additional CL domain.

59. The antibody or fragment thereof according to claim 57 or 58, wherein an additional CH1 domain is a wild-type CH1 domain and an additional CL domain is a wild-type CL domain.

60. The antibody or fragment thereof according to any one of claims 57 to 59, wherein the antibody or fragment thereof is selected from the group consisting of an asymmetric antibody and a F(ab’)2 fragment.61 . A fusion protein comprising the antibody or fragment thereof according to any one of claims 57 to 60.

62. The fusion protein according to claim 61 , wherein a peptide is fused to the N- and / or C-terminus of the antibody or fragment thereof.

63. The fusion protein according to claim 61 or 62, wherein the fusion protein is selected from the group consisting of (preferably from the N- to the C-terminus) asymmetric antibody-peptide2 and F(ab’)2-peptide2.

64. The fusion protein according to claim 62 or 63, wherein the peptide is selected from the group consisting of a cytokine, a receptor, a ligand, scFv and VHH.

65. The fusion protein according to any one of claims 61 to 64, wherein the fusion protein is selected from the group consisting of asymmetric antibody-cytokine2, asymmetric antibody- SCFV2, asymmetric antibody-VHH2, F(ab’)2-cytokine2, F(ab’)2-scFv2, and F(ab’)2-VHH2.

66. A mixture of at least two antibodies and / or fragments thereof and / or fusion proteins comprising an antibody or fragment thereof, wherein a first antibody or fragment thereof is the antibody or fragment thereof according to any one of claims 38 to 51 or a first fusion protein is the fusion protein according to any one of claims 52 to 56 [wherein each CH1 domain and each CL domain of the corresponding antibody or fragment thereof or fusion protein is (i) and (ii) of any one of claims 38 to 49]; and wherein each CH1 domain and each CL domain of each additional antibody or fragment thereof or additional fusion protein comprises a CH 1 domain different from (i) of any one of claims 38 to 49 and a CL domain different from (ii) of any one of claims 38 to 49, preferably wherein the mixture comprises two antibodies and / or fragments thereof and / or fusion proteins.

67. The mixture according to claim 66, wherein each additional antibody or fragment thereof or fusion protein comprises a CH1 domain matching a CL domain comprised in the additional antibody or fragment thereof or fusion protein.

68. The mixture according to claim 66 or 67, wherein an additional antibody or fragment thereof or fusion protein comprises a wild-type CH1 domain and a wild-type CL domain.

69. At least one nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 38 to 51 and 57 to 60, the fusion protein according to any one of claims 52 to 56 and 61 to 65, or the mixture according to any one of claims 66 to 68.

70. The at least one nucleic acid according to claim 69, wherein the nucleic acid is DNA or RNA, preferably mRNA.71 . A composition comprising (i) the antibody or antigen-binding fragment thereof according to any of claims 38 to 51 and 57 to 60, (ii) the fusion protein according to any of claims 52 to 56 and 61 to 65, (iii) the mixture according to any of claims 66 to 68, or (iv) the at least one nucleic acid according to claim 69 or 70.

72. The composition according to claim 71 , wherein the composition is a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.

73. The composition according to claim 71 or 72 for use in therapy.

74. The composition according to any one of claims 71 to 73 for use in the treatment of cancer.

75. The composition according to any one of claims 71 to 73 for use in the treatment of an infectious disease.

76. The composition according to any one of claims 71 to 73 for use in the treatment of an autoimmune disease.

77. The composition according to claim 71 , wherein the composition is a diagnostic composition.

78. Use of (i) the antibody or antigen-binding fragment thereof according to any of claims 38 to 51 and 57 to 60, (ii) the fusion protein according to any of claims 52 to 56 and 61 to 65, or (iii) the mixture according to any of claims 66 to 68 in diagnosis.

79. A method for the preparation of an antibody or fragment thereof according to any one of claims 38 to 51 and 57 to 60 or of a fusion protein according to any one of claims 52 to 56 and 61 to 65, wherein the method comprises the steps of(i) transforming a host cell with at least one nucleic acid encoding the antibody or fragment thereof or the fusion protein;(ii) culturing the host cell under conditions allowing the synthesis of the antibody or fragment thereof or the fusion protein; and(iii) recovering the antibody or fragment thereof or the fusion protein from the culture.

80. A method for the preparation of a mixture according to any of claims 66 to 68, wherein the method comprises the steps of(i) transforming a host cell with at least one nucleic acid encoding the mixture;(ii) culturing the host cell under conditions allowing the synthesis of the mixture; and(iii) recovering the mixture from the culture.

81. Use of the following substitutions in a constant heavy chain 1 (CH1) domain and a constant light chain (CL) domain to induce a preferential binding between the respective CH1 domain and the respective CL domain:(i) the amino acid substitutions S134C and C233V in the numbering according to Kabat corresponding to S136C and C220V in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S114C and C214V in the numbering according to Kabat corresponding to S114C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) the amino acid substitutions S134C, L143D, K145S and C233V in the numbering according to Kabat corresponding to S136C, L145D, K147S and C220V in thenumbering according to EU in the CH1 domain, and (a)(ii) the amino acid substitutions S114C, S131 R and C214V in the numbering according to Kabat corresponding to S114C, S131 R and C214V in the numbering according to EU in the CL domain; or (b)(i) the amino acid substitutions S134C, H172D and C233V in the numbering according to Kabat corresponding to S136C, H168D and C220V in the numbering according to EU in the CH1 domain, and (b)(ii) the amino acid substitutions S114C, L135K, N137K and C214V in the numbering according to Kabat corresponding to S114C, L135K, N137K and C214V in the numbering according to EU in the CL domain;(i) the amino acid substitutions A139Y, L143D and K145S in the numbering according to Kabat corresponding to A141Y, L145D and K147S in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F116A, S131 R and N137V in the numbering according to Kabat corresponding to F116A, S131 R and N137V in the numbering according to EU in the CL domain;(i) the amino acid substitutions S130M, G141Wand H172D in the numbering according to Kabat corresponding to S134M, G143W and H168D in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A, N137Q and D167K in the numbering according to Kabat corresponding to F118A, N137Q and D167K in the numbering according to EU in the CL domain;(i) the amino acid substitutions L124I, G141W, F174S and S188A in the numbering according to Kabat corresponding to L128I, G143W, F170S and S183A in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118G and S176F in the numbering according to Kabat corresponding to F118G and S176F in the numbering according to EU in the CL domain;(i) the amino acid substitutions (i’) L124I, G141W and K221 E in the numbering according to Kabat corresponding to L128I, G143W, and K213E in the numbering according to EU or (i”) L124I, G141 F and K221 D in the numbering according to Kabat corresponding to L128I, G143F and K213D in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118G, E123K and S131 D in the numbering according to Kabat corresponding to F118G, E123K and S131 D in the numbering according to EU in the CL domain;(i) the amino acid substitutions S130M, L143D, K145S and V190Y in the numbering according to Kabat corresponding to S134M, L145D, K147S and V185Y in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A and S131 R in the numbering according to Kabat corresponding to F118A and S131 R in the numbering according to EU in the CL domain;(i) the amino acid substitutions K145S, F174S, S186E and S188A in the numbering according to Kabat corresponding to K147S, F170S, S181 E and S183A in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S131 R and S176F in the numbering according to Kabat corresponding to S131 R and S176F in the numbering according to EU in the CL domain;(i) the amino acid substitutions F174C and C233V in the numbering according to Kabat corresponding to F170C and C220V in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions S162C and C214V in the numbering according to Kabat corresponding to S162C and C214V in the numbering according to EU in the CL domain; preferably (a)(i) the amino acid substitutions L143D, K145S, F174C and C233V in the numbering according to Kabat corresponding to L145D, K147S, F170C and C220V in the numbering according to EU in the CH1 domain, and (a)(ii) the amino acid substitutions S131 R, S162C and C214V in the numbering according to Kabat corresponding to S131 R, S162C and C214V in the numbering according to EU in the CL domain;(i) the amino acid substitutions (i’) T137W, T173V and V190W in the numbering according to Kabat corresponding to T 139W, T 169V and V185W in the numbering according to EU or (i”) G141 Wand K222R in the numbering according to Kabat corresponding to G143W and K214R in the numbering according to EU or (i’”) A125M, S127I and V190W in the numbering according to Kabat corresponding to A129M, S1311 and V185W in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions F118A, V133G and S174T in the numbering according to Kabat corresponding to F118A, V133G and S174T in the numbering according to EU in the CL domain; or(i) the amino acid substitutions H172Y and K221 E in the numbering according to Kabat corresponding to H168Y and K213E in the numbering according to EU in the CH1 domain, and (ii) the amino acid substitutions E123K, N137L, T164M and T180W in the numbering according to Kabat corresponding to E123K, N137L, T164M and T180W in the numbering according to EU in the CL domain.

82. The use according to claim 81 , wherein the CH1 domain and the CL domain are comprised in an antibody or a fragment thereof or a fusion protein comprising said antibody or fragment thereof.

83. An in vitro method of determining the percent of correctly paired heavy and light chains amongst paired heavy and light chains derived from at least two antibodies directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising at least (i) a heavy chain and a light chain of a first antibody and (ii) a light chain of a second antibody; b) determining the total amount of antibody by staining the paired heavy and light chains with an anti-Fc antibody and quantifying the signal of the anti-Fc antibody to arrive at a quantity 1 ; c) determining the amount of the first antibody by staining the paired heavy and light chains with an anti-idiotype antibody directed to the first antibody, wherein said anti-idiotype antibody binds only to correctly assembled heavy and light chains of the first antibody, andquantifying the signal of said anti-idiotype antibody to arrive at a quantity 2a; d) calculating a result according to the formula [quantity 2a I quantity 1] x 100; wherein the result indicates the percent of correctly paired heavy and light chains.

84. The method according to claim 83, wherein the determination steps b) and c) are carried out using an enzyme-linked immunosorbent assay (ELISA), preferably (i) wherein the determination step b) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reporter moiety-coupled coupled anti-human IgG-Fcy antibody as detection agent; and (ii) the determination step c) is carried out using an anchoring moiety-coupled anti-human IgG-Fc antibody as capture reagent and a reported moiety-coupled anti-idiotype antibody directed to the first antibody as detection agent.

85. An in vitro method of detecting mispaired heavy and light chains amongst paired heavy and light chains derived from at least one IgG antibody and at least one Fab fragment directed to different epitopes, wherein the method comprises the following steps: a) providing paired heavy and light chains comprising (i) a heavy chain and a light chain of an IgG antibody, wherein the heavy chain is not fused to a tag and the light chain is fused to a tag1 , and (ii) a VH-CH1-fusion chain and a light chain of a Fab fragment, wherein the VH-CH1 -fusion chain is not fused to a tag and the light chain is fused to a tag2; b) denaturing the paired heavy and light chains provided in step a) under nonreducing conditions; c) separating the denatured paired heavy and light chains according to the molecular weight; and d) staining the paired heavy and light chains separated according to molecular weight with (i) a detection agent specific for tag1 , (ii) a detection agent specific for tag2; and optionally (iii) a detection agent specific for human Fd; wherein the staining of the different detection agents can be discriminated from each other; wherein tag1 has a molecular weight of MW1 in kDa and tag2 has a molecular weight of MW2 in kDa, and wherein the difference between MW1 and MW2 is such that a discrimination between tag1 and tag2 is possible when separated according to the molecular weight; and wherein mispaired heavy and light chains are identified via their molecular weight and stain; and optionally wherein the ratio of (i) mispaired heavy and light chains to (ii) correctly pairedheavy and light chains and single chains is identified via the Fd staining.

86. The method according to claim 85, wherein (i) paired heavy and light chains of a molecular weight of about (50 kDa + MW1) stained with the agent specific for tagl ; (ii) paired heavy and light chains of about (75 kDa + MW2) stained with the agent specific for tag2; (iii) paired heavy and light chains of about (150 kDa + MW1 + MW2) stained with the agent specific for tag1 and the agent specific for tag2; and (iv) paired heavy and light chains of about (150 kDa + MW2 + MW2) stained with the agent specific for tag2 correspond to mispaired antibody chains.

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