WO2016102588A1 - Method for the production of antibodies - Google Patents

Abstract

The present invention relates to methods for the production of antibodies, for the screening and selection of antibodies with advantageous properties relative to other antibodies of a pool of a high number of antibodies.

Description

METHOD FOR THE PRODUCTION OF ANTIBODIES FIELD OF THE INVENTION

The present invention relates to methods for the screening and selection of antibodies.

BACKGROUND OF THE INVENTION Antibodies have in recent years become successful therapeutic molecules, in particular for the treatment of cancer and autoimmune diseases. To generate antibodies for therapeutic and other uses, several technologies exist and these have been reviewed by Strohl and Strohl (Therapeutic Antibody Engineering, Chapter 4, Woodhead Publishing Limited, 2012).

Hybridoma technology, first described by Kolher and Millstein in 1975 ("Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256: 495-7) was the first technology available to generate monoclonal antibodies. Hybridomas are generated by fusion of antibody producing B cells to an immortal cell line. By limiting dilution or other ways of generating single cell-derived clones, an infinite source of a specific monoclonal antibody is made. This technology a.o. can be used to obtain monoclonal antibodies from rodents and rabbits, either wild type or transgenic animals that express (parts of) the human antibody repertoire. Most often these animals have been immunized with the antigen of interest.

Different display technologies can be used for the generation and screening of libraries of antibody gene sequences (Strohl and Strohl 2012, supra; Hoogenboom 2005 "Selecting and screening recombinant antibody libraries", Nature Biotechnolology, 23: 1105-16). The variety of display technologies includes phage, yeast, bacterial, ribosome and mRNA, and

mammalian cell display. For these, the library of antibody gene sequences can be obtained from naive or immunized animals or humans, but can also be synthetically constructed. The antibody repertoire is displayed on individual phages, yeast particles etc, followed by selection or screening to identify antibody sequences that bind to the target antigen.

Subsequently, the nucleic acid sequences are amplified, with or without further introduction of diversity. The entire screening and selection is done on the display particles or cells and is solely based on binding, only after amplification of the DNA sequences are soluble antibodies produced that can be used in further functional characterization.

It is now found that a technology could be developed based on the generation and combination of pools of antibody heavy- (HC) and light chain (LC) sequences that allows for direct screening for the most optimal heavy and light chain combination(s) in a soluble format. This technology can be applied to any source of antibody sequence.

OBJECT OF THE INVENTION

It is an object of the invention to provide methods for selecting and screening for antibodies with advantageous properties in terms of specificity, binding affinity, or with other improved or advantageous functional characteristics or properties like internalization, inhibition of signalling, and other antibody mediated effector functions.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that unique antibodies may be provided and selected out of a large amount LC and HC combinatorial possibilities by applying a pool approach as described herein.

In particular the present invention relates to the pairing of LC and HC immunoglobulin molecules that may not have been combined if the antibody had been produced in traditional ways and direct selection and screening of the resulting antibodies in soluble form. So, in a first aspect the present invention relates to a method for the selection of an individual antibody, the antibody binding at least one antigen of interest and comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Co-transfecting cells with each one individual clone expressing a specific HC of said HC pool together with said LC pool comprising a plurality of individual clones expressing LC to express antibodies from said cells consisting of said specific HC randomly combined with LC(s) from said LC pool, this co-transfection being performed for each individual clone of said HC pool; iii) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual HC clones; iv) Co-transfecting cells with the clone(s) expressing each HC selected under step iii) together with each one individual clone expressing a specific LC of said LC pool to express antibodies with combined HC and LC; and v) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said HC selected under step iii) to provide the best combined LC and HC antibody.

In a second aspect the present invention relates to a method for the selection of an individual antibody, the antibody binding an antigen of interest and comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Co-transfecting cells with each one individual clone expressing a specific LC of said LC pool together with said HC pool comprising a plurality of individual clones expressing HC to express antibodies from said cells consisting of said specific LC randomly combined with HC(s) from said HC pool, this co-transfection being performed for each individual clone of said LC pool; iii) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual LC clones; iv) Co-transfecting cells with the clones expressing each LC selected under step iii) together with each one individual clone expressing a specific HC of said HC pool to express antibodies with combined LC and HC; and v) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said LC selected under step iii) to provide the best combined LC and HC antibody.

In a further aspect the present invention relates to a method for the production of a recombinant antibody binding an antigen or antigens of interest and comprising a heavy chain (HC) and a light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Co-transfecting cells with each one specific clone expressing a specific HC of said HC pool together with a composition comprising all clones of said LC pool to express antibodies from said cells consisting of said specific HC randomly combined with LC(s) from said LC pool, this co-transfection being performed for each individual clone of said HC pool; iii) Screening for and selecting the best HC of said antibodies obtained under step ii) to provide individual HC clones; iv) Co-transfecting cells with clones expressing each HC selected under step iii) together with each one specific clone expressing a specific LC of said LC pool to express antibodies with combined HC and LC; v) Screening for and selecting the best LC of said antibodies obtained under step iv) when in combination with said HC selected under step iii) to provide the best combined LC and HC antibody; and vi) Inserting the nucleic acids encoding the said selected antibody from v) in a host cell, expressing the antibody, isolating and purifying the antibody.

In a further aspect the present invention relates to a method for the production of a recombinant antibody binding an antigen or antigens of interest and comprising a heavy chain (HC) and a light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing

HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Co-transfecting cells with each one specific clone expressing a specific LC of said LC pool together with a composition comprising all clones of said HC pool to express antibodies from said cells consisting of said specific LC randomly combined with HC(s) from said HC pool, this co-transfection being performed for each individual clone of said LC pool; iii) Screening for and selecting the best LC of said antibodies obtained under step ii) to provide individual LC clones; iv) Co-transfecting cells with clones expressing each LC selected under step iii) together with each one specific clone expressing a specific HC of said HC pool to express antibodies with combined LC and HC; v) Screening for and selecting the best HC of said antibodies obtained under step iv) when in combination with said LC selected under step iii) to provide the best combined HC and LC antibody; and vi) Inserting the nucleic acids encoding the said selected antibody from v) in a host cell, expressing the antibody, isolating and purifying the antibody.

In a further aspect the present invention relates to an antibody obtainable by or obtained by the method according to the invention.

LEGENDS TO THE FIGURE

Figure 1; Typical example of a LC screen; 4 ELISA plates are shown, each coated with the AxlECDHis protein. In each separate plate a single HC combined with 94 individual single LCs is tested. The top left corner well is a negative control, the second well in the first column is a Axl binding positive control antibody. Reactivity of antibodies with the coated AxlECDHis protein is represented as dark coloring of the well. As can be seen each HC can combine with at least 6 LCs out of 94 (the 2 left plates) or more (2 right plates)

Figure 2: Example of average binding characterization of antibodies derived from mouse 552934. Figure 2A depicts the total number of sequences beloning to each HC CDR3 cluster. Binders were identified based on the number of objects above background and depicted in black. The number of non-binding samples is indicated in white. The background number of objects was established using negative control antibodies. Figure 2B shows the average total binding intensity to CHO-SORT for the samples identified as binders (black) and the samples identified as non-binders (white) are plotted. Cluster -1 contain all HC sequences that failed to give a good sequence reads. The number of objects identified for negative control antibodies was≤50. Samples were considered binders when the number of objects (NO) >50.

Figure 3: Figure 3A depicts the number of LC sequences in a LC cluster that are identified as binder (black NO > 50) or non-binder (white; NO≤50). Figure 3B depicts the average binding signal of all binding LC in a LC CDR3 cluster (black) and all non-binding LCs in a LC cluster (white). DETAILED DISCLOSURE OF THE INVENTION Definitions

The term "antibody" as used herein is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The binding region (or binding domain which may be used herein, both having the same meaning) which interacts with an antigen, comprises variable regions of the heavy and/or light chains of the immunoglobulin molecule. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically interact, such as bind, to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_H1 domains, or a monovalent antibody as described in WO2007059782 (Genmab A/S); (ii) F(ab')₂ fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting essentially of the V_H and C_H1 domains; (iv) a Fv fragment consisting essentially of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a V_H domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003

Nov;21(ll) :484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(l) : 111-24) and (vii) an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,

5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

The term "immunoglobulin heavy chain" (HC) or "heavy chain of an immunoglobulin" or just "heavy chain" as used herein is intended to refer to one of the heavy chains of an

immunoglobulin. Similarly the term "immunoglobulin light chain" (LC) or "light chain of an immunoglobulin" or just "light chain" as used herein is intended to refer to one of the light chains of an immunoglobulin. A heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light low molecular weight chains (LC) and one pair of heavy chains (HC), all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized (see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Within the structure of the immunoglobulin, the two heavy chains are inter-connected via disulfide bonds in the so- called "hinge region". Equally to the heavy chains each light chain is typically comprised of several regions; a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. Furthermore, the VH and VL regions may be further subdivided into regions of

hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4 (see also Lefranc MP et al, Dev Comp Immunol Jan: 27(l): 55-77 (2003)). The term "full-length antibody" when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and/or light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype. The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be produced by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

The term "antigen-binding region" as used herein, refers to a region of an antibody which is capable of binding to the antigen. The antigen can be any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virus.

The term "binding" as used herein refers to the binding of an antibody to a predetermined antigen or target, typically with an affinity corresponding to a K_D of about 10^"5 M or less, e.g. 10^"7 M or less, such as about 10^"8 M or less, such as about 10^"9 M or less, about 10 ¹⁰ M or less, or about 10 ¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the protein as the analyte, and binds to the predetermined antigen with an affinity corresponding to a K_D that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the K_D of the protein, so that when the K_D of the protein is very low (that is, the protein is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.

The term "k_d" (sec ¹), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the k_0ff value.

The term "k_a" (M ¹ x sec^"1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.

The term "K_D" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. The term "K_A" (M ¹), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the k_a by the k_d.

The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in this context still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody and in some cases this may be associated with greater affinity, selectivity and/or specificity than the parent antibody. Such functional variants typically retain significant sequence identity to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, which is well-known in the art.

The VH, VL and/or CDR sequences of variants may differ from those of the parent antibody sequences through mostly conservative substitutions; for instance at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements.

The VH, VL and/or CDR sequences of variants may differ from those of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

The term "amino acid" and "amino acid residue" may herein be used interchangeably, and are not to be understood limiting.

It is well-known within the art when an amino acid sequence comprises an "X" or "Xaa", said X or Xaa represents any amino acid. Thus, X or Xaa may typically represent any of the 20 naturally occurring amino acids. The term "naturally occurring" as used herein refers to any one of the following amino acid residues; glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine.

The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of 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. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues which are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).

The term "target(s)" as used herein refers to a molecule to which the binding region of the protein according to the invention binds. When used in the context of the binding of an antibody includes any antigen towards which the raised antibody is directed. The term "antigen(s)" and "target(s)" may in relation to the invention be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. It is to be understood that an antibody according to the invention may bind more than one antigen of interest, such as in case of cross reactive antibodies or with closely related antigens. It is also understood that included with the term "antigen" is parts of an antigen, such as where immunizations for an antigen are made with specific domains of that antigen to focus an immune response to that particular region of the antigen.

The term "pool" as used herein refers to a plurality of individual clones that can be characterized based on any type of antibody/binding molecule characteristic. Accordingly an LC pool is a plurality of individual clones each expressing immunoglobulin light chain molecules upon transfection in a host cell.

The term "clone" or "clones" as used herein refers to any individual piece of nucleic acid sequence that may express an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, or another antigen binding molecule such as a T cell receptor. The term may refer to genomic DNA, cDNA, RNA and may be derived from any suitable source such as a synthetic library, genomic DNA, protein sequence information, a cDNA library, or a T cell receptor library.

The term "plurality" refers to an amount of 2 or more, such as more than, 2, 3, 4, 5 6, 7, 8, 9, 10, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000.

In some embodiments the term plurality refers to an amount of 2 or more, such as more than 3, 4, 5, 6, 7, 8, 9, 10, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000, with an upper limit of 2 or more, such as 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000.

The term "at least one antigen" refers to one antigen or multiple antigens of interest, e.g. one antigen if both HC(s) and LC(s) binds the same and only one antigen, or it may refer to two or more antigens if e.g. the HC(s) and LC(s) bind different antigens, or if the antibody just cross-reacts with more than one antigen of interest.

The methods according to the present invention have many advantages over current methods for providing antibodies including hybridoma technology. These include:

- Flexibility: after each step, samples can be stored and the method can be continued when needed.

- After the first step, samples are secured (no loss of samples after contamination, always possible to start from previous step).

- The source material can include cells that are not suited for traditional hybridoma technology (memory cells, plasma cells) as well as tissues.

- The antibody format which is favoured for a specific target and/ or application can be chosen (by cloning into the desired expression vectors, with the desired antibody constant regions) at the start of the screening and selection process, enabling screening and selection based on the specifics of the format. - Sequence information on the full antibody library is available at an early stage enabling additional selection or omission based on sequence characteristics, the selection of additional antibodies based on sequence homology as identifying critical residues in antibody clusters based on binding and non-binding data.

- Binding data of a large panel of HC and LC combined Abs, delivers a range of affinities of similar Abs, enabling to study the effects of affinity on Ab function.

It is an advantage of the present invention that the traditional hybridoma step can be left out when the methods according to the present invention are used, such as when animals, such as transgenic mice, rabbits or rats, are used for generating antibodies or HC pools or LC pools. This may provide for a faster and more efficient method of generating a plurality of antibodies for screening and selection of antibodies with improved properties.

In some aspects according to the present invention, it is desirable to reduce a pool size before screening individual clones. This may be accomplished by screening e.g. pools of LCs against pools of HCs. This is further illustrated in the below examples: In one example we have a source of Ab expressing cells in which 1/1000 cells contains an unique binding HC and in which 1/1000 cells contains a unique binding LC, which provides for a unique HC/LC combo.

To find this unique combo one may do this, for example.

1. Screen all individual clones in a matrix lOOOx 1000 = le6 (1x10^s) transfections/ assays. 2. Divide clones into 10 pool of 100 HCs and 100 LCs each. First screen 10x10 pools= 100 assays, identify positive pools and screen individual clones from these pools, lOOx 100= 10000. In total 2 screens, 10100 assays.

2a. Or alternatively, divide positive pools from the 1^st screen in 10 pools of 10 and do a 2^nd screen 10x10 pools= 100 assays, identify positive pools and screen individual clones of the positive 2^nd screen pools = 10x10= 100 in a 3^rd screen. Total 3 screens, 300 assays.

3. or asymmetrically 100 pools of 10 HCs x 10 pools of 100 LCs = 1000 assays. 2^nd screen individual HCs vs individual LCs from the positive pools = 10x100= 1000 assays. In total 2 screens, 2000 assays. The specific number of pools and division into smaller numbers may be varied . The main effect/ strength of pooling HCs and/or LCs is to drastically reduce the transfection/ assay numbers. The best strategy may be dictated by and adaptable to the antibody source, capacity, expression levels, the detection limit of the assay and practical considerations. The immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC) are in the methods according to the present invention selected based on the screening for some pre-set criteria. These may include qualitative (specific binding or not), or it may be quantitative. In some specific embodiments, the pre-set criterion is qualitative binding or not as determined in the assay described as "Qualitative binding assay" in the examples. Alternative pre-set criteria may include qualitative or quantitative measure of internalization, inhibition of signalling, and other antibody mediated effector functions. Assays for measurement of such characteristics may include the following :

Methods to screen for functional characteristics of antibodies include amongst others methods to measure antibody internalization (such as described by Poul et al J . Mol . Biol . (2000) 301, 1149-1161, Lammerts van Bueren et al, Cancer Res 2006; 66: 7630-7639, Liu et al, Cancer Biother Radiopharm (2007) ; 22: 33-9, De Goeij et al, mAbs 2014; 6: 2, 392- 402), target down-modulation (for instance as described by De Goeij et al, mAbs 2014; 6: 2, 392-402), inhibition or enhancement of signaling (such as described by Krutzik and Nolan, Nature Methods (2006) 3, 361-368, Bleeker et al, Journal of Immunology (2004), 173 :

4699-4707), inhibition or enhancement of ligand/receptor interactions (such as described by Bleeker et al, Journal of Immunology (2004), 173 : 4699-4707, Schaefer et al Cancer Cell (2011) 20, 472-486), influence on cell growth (such as in Schaefer et al Cancer Cell (2011) 20, 472-486, apoptosis (such as described by Vermes et al Journal of Immunological Methods 184 (1995) 39-51, Otsuki, Acta Histochem Cytochem (2000) 33, 235-241) or antibody mediated effector functions (such as in Teeling et al, Blood (2004) 104, 1793-1800, Bleeker et al, Journal of Immunology (2004), 173 : 4699-4707, Gerritsen et al, Journal of Immunological Methods 352 (2010) 140- 146, Schnueriger et al, Molecular Immunology 48 (2011) 1512- 1517) .

Specific embodim ents of the invention

As described above the present invention relates to a method for the selection of an individual antibody, the antibody binding at least one antigen of interest and comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing

HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Co-transfecting cells with each one individual clone expressing a specific HC of said HC pool together with said LC pool comprising a plurality of individual clones expressing LC to express antibodies from said cells consisting of said specific HC randomly combined with LC(s) from said LC pool, this co-transfection being performed for each individual clone of said HC pool; iii) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual HC clones; iv) Co-transfecting cells with the clone(s) expressing each HC selected under step iii) together with each one individual clone expressing a specific LC of said LC pool to express antibodies with combined HC and LC; and v) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said HC selected under step iii) to provide the best combined LC and HC antibody.

In an alternative aspect in the method, HC is just replaced with LC and vice versa.

That is in another aspect of the invention the method for the selection of an individual antibody, the antibody binding at least one antigen of interest and comprising an

immunoglobulin light chain (LC) and an immunoglobulin heavy chain (HC), which method comprises the steps of i) Providing a first LC pool comprising a plurality of individual clones expressing LC and a first HC pool comprising a plurality of individual clones expressing HC; ii) Co-transfecting cells with each one individual clone expressing a specific LC of said LC pool together with said HC pool comprising a plurality of individual clones expressing HC to express antibodies from said cells consisting of said specific LC randomly combined with HC(s) from said HC pool, this co-transfection being performed for each individual clone of said LC pool; iii) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual LC clones; iv) Co-transfecting cells with the clone(s) expressing each LC selected under step iii) together with each one individual clone expressing a specific HC of said HC pool to express antibodies with combined LC and HC; and v) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said LC selected under step iii) to provide the best combined HC and LC antibody.

It is to be understood that the pools of HC and of LC may be provided from any suitable source as this is not essential to the invention. The co-transfection under step ii) are to be performed with each single clone of either HC or LC in combination with the entire pool of LC or HC respectively. Accordingly, either the HC or the LC clone is fixed for each transfection and combined with the entire pool of LC or HC respectively. In step iv) are individual clones of both LC and HC co-transfected in order to determine the specific advantageous LC/HC combination.

In some embodiments the method according to the present invention comprises the preceding steps of a) Providing a plurality of individual clones expressing HC in one or more first HC pools and a plurality of individual clones expressing LC in one or more first LC pools; b) Co-transfecting cells with each of said first HC pool together with each of said first LC pool to express antibodies from said cells consisting of randomly combined HC and LC from said first HC and LC pools, this co-transfection being performed for each individual first HC pool and/or for each individual first LC pool; c) Screening for and selecting the HC pools and/or the LC pools based on pre-set criteria of said antibodies obtained under step b) to provide one or more second HC pools and/or one or more second LC pools; d) Using the pools obtained under step c) in step i) of claims 1 or 2. That is the one or more second HC pools and/or second LC pools can be used in step i) described above as the first HC pool and/or first LC pool. It is to be understood that these steps a)-d) may precede the method steps i)-v) described above. In this embodiment, pool sizes may be reduced significantly by screening pools of HC against pools of LC. Accordingly, the pools entering the method steps i)-v) are reduced in number of individual clones contained within a particular pool.

In some embodiments in the method according to the present invention steps a)-c) are repeated one or more times for the selected HC pools and/or LC pools so that said second pools selected under step c) becomes said first LC pools and/or HC pools of step a) before proceeding with step d).

In some embodiments the method according to the present invention comprises the preceding steps of i) isolating RNA comprising RNA encoding the heavy chain of antibodies from each of a plurality of antibody expressing cells derived from a plurality of antibody producing individuals such as naive individuals or individuals immunized with said same antigen of interest; ii) preparing cDNA of said RNA encoding heavy chains (HC) of said antibodies and cloning the cDNA into an expression system; iii) selecting individual clones from step b) to prepare a plurality of individual clones expressing HC (HC pool); and iv) using the pools obtained under step c) in step i) of claims 1-2 or in step a) of claims 3-4. That is the HC pool(s) can be used in step i) described above as the first HC pool and/or it can be used in step a) described above as the first HC pool(s).

In some embodiments the method according to the present invention comprises the preceding steps of i) isolating RNA comprising RNA encoding the light chain of antibodies from each of a plurality of antibody expressing cells derived from a plurality of antibody producing individuals, such as naive or individuals immunized with said same antigen of interest; ii) preparing cDNA of said RNA encoding light chains (LC) of said antibodies and cloning the cDNA into an expression system; and iii) selecting individual clones from step b) to prepare a plurality of individual clones expressing LC (LC pool); and iv) using the pools obtained under step c) in step i) of claims 1-2 or in step a) of claims 3-4. That is the LC pool(s) can be used in step i) described above as the first LC pool and/or it can be used in step a) described above as the first LC pool(s).

In some embodiments in the method according to the present invention the plurality of antibody expressing cells are B lymphocytes or spleen cells obtained directly from said individual.

In some embodiments in the method according to the present invention steps i)-iii) and optionally steps iv)-v) are repeated under different pre-set criteria for the selected HCs so that the one or more individual HC clones provided under step iii) and/or step v) becomes the LC pool and/or HC pool of step i).

In some embodiments of the method according to the present invention steps a)-c) are repeated under different pre-set criteria for the selected HCs and/or LCs, so that the one or more individual HC or LC clones provided under step c) becomes the first HC and/or LC pool of step a).

In some embodiments in the method according to the present invention the HC and the LC bind the same antigen.

In some embodiments in the method according to the present invention the HC and the LC bind different antigens.

In some embodiments in the method according to the present invention the plurality of individual clones expressing HC (HC pool) and/or the plurality of individual clones expressing LC (LC pool) is obtained from a synthetic library, genomic DNA, protein sequence

information, a cDNA library, T cell receptor library, or from the method steps described in claims 3-4 (that is the plurality of individual clones expressing HC or LC can be obtained as described herein).

In some embodiments in the method according to the present invention the plurality of individual clones expressing HC (HC pool) and/or the plurality of individual clones expressing LC (LC pool) derives from antibody producing cells, such as cells of the blood circulation, bone marrow cells, spleen cells, such as splenocytes of an animal, such as an immunized animal or naive animal, such as mouse, rabbit, rat, guinea pig, or camel. In some embodiments the method according to the present invention comprises a step of preparation of Linear expression elements (LEE's) encoding the HC and/or LC.

In some embodiments in the method according to the present invention the first and/or the second LC pool consist of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing LC. In some embodiments in the method according to the present invention the first and/or the second HC pool consist of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing HC.

It is to be understood that the pool size may be reduced in size through steps of screening pools of LC against pools of HC and accordingly, the specific size, i.e. the amount of individual clones expressing HC or LC may change, such as be reduced through the method. In some embodiments in the method according to the present invention the first and/or second HC pool and/or the first and/or second LC pool derives from at least 2, 3, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 2-100, 10-100, 20-100, 30-100, 40-100, 50-100, or 60-100 individual animals, such as mouse, rabbit, rat, guinea pig, or camel, naive or immunized with the same or different specific antigen.

In some embodiments in the method according to the present invention the primers specific for the amplification of IgM or IgGl HC VHs are used under step b) in the amplification of HC encoding cDNA molecules.

In some embodiments in the method according to the present invention the primers specific for the amplification of kappa or lambda LCs are used under step b) in the amplification of LC encoding cDNA molecules. In some embodiments in the method according to the present invention the pre-set criteria for screening and selecting the HCs under step iii) are based on specific binding, such as in a qualitative binding assay determining binding to a specific antigen.

In some embodiments in the method according to the present invention the pre-set criteria for screening and selecting the LCs under step iii) are based on specific binding, such as in a qualitative binding assay determining binding to a specific antigen.

In some embodiments in the method according to the present invention the first and/or second HC pool and/or the first and/or second LC pool derives from a transgenic animal, such as mouse, rabbit, rat, camelid immunized to produce fully human or humanized antibodies or from sequence information of fully human or humanized antibodies.

In some embodiments in the method according to the present invention under step b) VH and VL coding regions are amplified by 5' RACE or PCR before cloning into an expression system .

In some embodiments in the method according to the present invention under step b) VH and VL coding regions are cloned into a bacterial, yeast, plant, or mammalian expression vector.

In some embodiments in the method according to the present invention the pre-set criteria for screening and selecting the combined HC and LC antibody or antibodies in step v) is based on antigen binding ELISA, or homogeneous binding assay. In some embodiments the method according to the present invention screens for and selects antibodies with a dissociation constant (KD) lower than 10-6M, such as lower than 5xlO-7M, such as lower than 10-8M, such as lower than 5xlO-9M, , such as lower than 10-9M, such as lower than 5xl0-10M.

In some embodiments in the method according to the present invention the pre-set criteria for screening and selecting of the combined HC and LC antibody or antibodies in step v) is based on a functional assay screen such as but not limited to an internalization assay selecting antibodies inducing target internalization or lysosomal targeting or target down modulation, or signalling inhibition/activation or cell kill or target (cells) crosslinking.

In some embodiments the first and/or second LC pool consist of less than 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,

4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,

1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing LC.

In some embodiments the first and/or second HC pool consist of less than 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,

1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing HC.

EXAMPLE 1

Expression constructs for AXL

The following codon-optimized constructs for expression of various full-length AXL variants were generated : human (Hom o sapiens) AXL (Genbank accession no. NP_068713.2), human- cynomolgus monkey chimeric AXL in which the human extracellular domain (ECD) was replaced with the ECD of cynomolgus monkey (Macaca fascicularis) AXL (translation of Genbank accession HB387229.1 ; aa 1-447), human-mouse chimeric AXL in which the human ECD was replaced with the ECD of mouse (Mus m usculus) AXL (Genbank accession

NP_033491.2; aa 1-441), human-mouse chimeric AXL in which the human Ig-like domain I (aa 1-147, also termed "Ig l domain" herein) was replaced with the Ig-like domain I of mouse AXL, human-mouse chimeric AXL in which the human Ig-like domain II (aa 148-227, also termed "Ig2 domain" herein) was replaced by the Ig-like domain II of mouse AXL, human- mouse chimeric ALX in which the human FNIII-like domain I (aa 228-326, also termed "FN 1 domain" herein) was replaced with the FNIII-like domain I of mouse AXL, human-mouse chimeric AXL in which the human FNIII-like domain II (aa 327-447, also termed "FN2 domain" herein) was replaced by the FNIII-like domain II of mouse AXL. In addition, the following codon-optimized constructs for various AXL ECD variants were generated : the extracellular domain (ECD) of human AXL (aa 1-447) with a C-terminal His tag (AXLECDHis), the FNIII-like domain II of human AXL (aa 327-447) with a N-terminal signal peptide and a C-terminal His tag (AXL-FN2ECDHis), and the Ig l- and Ig2-like domains of human AXL (aa 1- 227) with a C-terminal His tag (AXL-Ig l2ECDHis) .

The constructs contained suitable restriction sites for cloning and an optimal Kozak

(GCCGCCACC) sequence (Kozak et al . (1999) Gene 234: 187-208) . The constructs were cloned in the mammalian expression vector pcDNA3.3 (Invitrogen) . AXL expression in EL4 cells

EL4 cells were stable transfected with the pcDNA3.3 vector containing the full length human AXL coding sequence and stable clones were selected after selection with the antibiotic agent, G418, (Geneticin).

Purification of His-taqqed AXL

AXLECDHis, AXL-FN2ECDHis, and AXL-Igl2ECDHis were expressed in HEK293F cells and purified with immobilized metal affinity chromatography.

Immunization

Material from 4 transgenic mice expressing human antibody gene sequences was used for selecting antibodies. Mice immunized with various immunization protocols and with various antibody responses and yielding various numbers of antibodies from the traditional hybridoma process were chosen. Mouse A (3.5 % hits in the hybridoma process) was a HCol7- BALB/c transgenic mouse (Bristol-Myers Squibb, Redwood City, CA, USA) was immunized alternatingly intraperitoneally (IP) with 20 μg AXL-FN2ECDHIS plus 20 μg AXL- Igl2ECDHis) and subcutaneously (SC) at the tail base) with the same protein, with an interval of 14 days. In total 8 immunizations were performed: 4 IP and 4 SC immunizations. For most immunizations, the first immunization was performed in complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) and all subsequent immunizations in incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). Mouse B (0 % hits in the hybridoma process) was a HCol2 transgenic mouse (Medarex) immunized with 20 μg of the AXLECDHis protein using a similar immunization protocol as mouse A. Mouse C (38 % hits in the hybridoma process) was a HCol2- BALB/c mouse immunized alternating intraperitoneally (IP) with EL4 cells transfected with full length human AXL in PBS and subcutaneously (SC; at the tail base) with the AXLECDHis protein in IFA, with an interval of 14 days. Mouse D (0 % hits in the hybridoma process) was a HCol2 transgenic mouse (Medarex) immunized with 20 μg of the AXL-Igl2ECDHis protein in using a similar immunization protocol as mouse A.

Mice with at least two sequential AXL specific antibody titers of 200 (serum dilutions of 1/200) or higher, were boosted 3-4 days prior to fusion (10 μg of AXL-derived protein in PBS injected intravenously).

Isolation of RNA from spleen cells

Total RNA was isolated from spleen cells using the Mini RNA easy kit (Qiagen). First strand cDNA for 5 '-RACE was synthesized using 150 ng of RNA using the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA), PrimeScript Reverse Transcriptase (Clontech) and the SMART HA oligo and oligodT as primers. VL encoding regions were amplified by PCR using Advantage 2 polymerase (Clontech), the primers RACEkLIC4shortFW2 (320 nM), RACEkLIC4LongFW2 (80 nM) and RACEkLICRV_PmlA3 (400 nM), performing 35 cycles of 30 seconds at 95 °C, and 1 minute at 68 °C. VH encoding regions were amplified by PCR using Pfu Ultra II Fusion HS DNA polymerase (Stratagene), the primers

RACEGl LIC3shortFW (320 nM), RACEGlLIC3longFW (80 nM) and RACEG1LIC3RV2 (400 nM), performing 40 cycles of 20 seconds at 95 °C, 20 seconds at 66 °C and 30 seconds at 72 °C, ending with a finale extension step of 3 minutes at 72 °C. VH or VL encoding PCR products were separated using agarose gel electrophoresis and DNA products of the expected size were cut from the gel and purified using the Qiagen MiniElute kit. VH and VL coding regions amplified by PCR were cloned, in frame, in the mammalian expression vectors pGlf

(containing the human IgGl constant region encoding DNA sequence) for the VH region and pKappa (containing the kappa light chain constant region encoding DNA sequence) for the VL region, by ligation independent cloning (Aslanidis, C. and P.J . de Jong, Nucleic Acids Res

1990; 18(20) : 6069-74) in E.coli strain DH5aTlR (Life technologies), yielding single bacterial colonies each containing a single HC or LC expression vector.

Table 1 : Primer sequences

Pri m er na m e Pri m e r seq ue n ce

SMARTIIA 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG

RACEkLIC4shortFW2 5'-ACGGACGGCAGGACCACT

RACEkLIC4LongFW2 5'-ACGGACGGCAGGACCACTAAGCAGTGGTATCAACGCAGA

RACEkLICRV PmlA3 5'-CAGCAGGCACACCACTGAGGCAGTTCCAGATTTC

RACEGl LIC3shortFW 5'-ACGGACGGCAGGACCAGT

RACEGlLIC3longFW 5'-ACGGACGGCAGGACCAGTAAGCAGTGGTATCAACGCAGAGT

RACEG1LIC3RV2 5'-GGAGGAGGGCGCCAGTGGGAAGACCGA

CMV P f (RRA2) 5'-GCCAGATATACGCGTTGACA

TK pA r (RRA2) 5'-GATCTGCTATGGCAGGGCCT

LEE PCR

Linear expression elements (LEE's) were produced by amplifying the fragment containing the CMV promoter, HC or LC encoding regions and the poly A signal containing elements from the expression plasmids. For this the regions were amplified using Accuprime Taq DNA polymerase (Life Technologies) and the primers CMVPf(BsaI)2 and TkpA(BsaI)r, performing 35 cycles of 45 seconds at 94 °C, 30 seconds at 55 °C and 2 (LC) or 3 (HC) minutes at 68 °C, using material of E.coli (strain DH5a) colonies, containing the plasmids, as a DNA template.

Transient expression in HEK-293 cells Antibodies were expressed as IgGl,K. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Freestyle 293-F (HEK293F) cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink, T., et al. (2014) (Ά simple, robust and highly efficient transient expression system for producing antibodies', Methods, 65 (1), 5-10)

For LEE expression of Abs 1 μΙ of the HC LEE PCR reaction mixture, 1 μΙ of the LC PCR reaction mixture and 1 μΙ of a 30 ng/ μΙ enhancing mix containing a mix of 3 expression enhancing plasmids as described in Vink, T., et al. (2014), were mixed and transfected in HEK293F cells in a total volume of 100 μΙ using 293 fectin as transfection reagent, according to the instructions of the manufacturer (Life technologies), using 96 well plates as vessel, essentially as described supra.

AXLECDHis ELISA

ELISA plates (Greiner, Netherlands) were coated with 100 μΙ / well of 0.5 μg/ ml AXLECDHis in Phosphate buffered saline (PBS) and incubated for 16 hours at room temperature (RT). The coating solution was removed and the wells were blocked by adding 150 μΙ PBSTC (PBS containing 0.1 % tween-20 and 2% chicken serum) well and incubating for 1 hour at RT. The plates were washed three times with 300 μΙ PBST (PBS containing 0.1 % tween-20)/well and 100 μΙ of test solution was added, followed by an incubation of 1 hour at RT. After washing three times with 300 μΙ of PBST/well, 100 μΙ antibody goat anti human IgG coupled with horse radish peroxidase (diluted 1/3000) was added and incubated for 1 hour at RT. After washing three times with 300 μΙ of PBST/well, 100 μΙ of ABTS (lmg/ml) solution was added and incubated at RT until sufficient signal was observed and the reaction was stopped by adding 100 μΙ of 2 % oxalic acid solution. 96 well plates were measured on an ELISA reader at 405 nm.

Diversity screen

Samples were analyzed for binding of antibodies to TH1021-hAXL (HEK293F cells transiently expressing the human AXL), TH1021-cAXL (HEK293F cells transiently expressing human- cynomolgus AXL chimeras in which the human ECD had been replaced with the ECD of cynomolgus monkey AXL), TH1021-mAXL (HEK293F cells transiently expressing human- mouse AXL chimeras in which the human ECD had been replaced with the ECD of mouse AXL), TH1021-mIgl (HEK293F cells transiently expressing the human AXL with the Ig-like domain I being replaced by the Ig-like domain I of mouse AXL), TH1021-mIg2 (HEK293F cells transiently expressing human AXL with the Ig-like domain II being replaced by the Ig- like domain II of mouse AXL), TH1021-mFNl (HEK293F cells transiently expressing human AXL with the FNIII-like domain I being replaced by the FNIII-like domain I of mouse AXL), TH1021-mFN2 (HEK293F cells transiently expressing human AXL with the FNIII-like domain II being replaced by the FNIII-like domain II of mouse AXL), and HEK293F cells (negative control which does not express AXL), respectively.

Samples from the LEE expression were added to the cells to allow binding to the various AXL constructs. Subsequently, binding of antibodies was detected using a fluorescent conjugate (Goat anti-Human IgG Fc gamma-DyLight649; Jackson ImmunoResearch). The samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and mean fluorescence was used as read-out. Samples were stated positive when counts were higher than 50 and counts x fluorescence was at least three times higher than the negative control.

Provision of HC and LC pools:

For each mouse, 352 HC expression vector containing bacterial colonies and 384 LC expression vector containing bacterial colonies were picked and amplified by LEE PCR. Part of the LEE reaction was sequenced (AGOWA). The percentage proper VH insert containing constructs differed largely between the 4 mice, mouse A (50 %), mouse B (23 %), mouse C (90 %) and mouse D (14 %) and resembled the variation of hits obtained in the hybridoma process, see supra. The HC diversity in the mice with only a limited amount of proper inserts were dominated by a large group of identical HCs, 65/83 in mouse B and 46/49 in mouse D. For mouse B and D the unique HCs (9 for mouse B, 4 for mouse D) were selected. For mouse A and C no selection was made.

Co-transfection of HCs with a LC pool.

The single HC encoding LEE's were co-transfected with a pool of 96 LC encoding LEE's using the LEE transfection protocol.

HC selection of Axl binding antibodies: For mouse B and D, supernatants from the LEE co-transfections of the single HC with the pooled LCs were analyzed for Axl binding of the produced antibody mixtures by the Axl ELISA. 7 of the 9 HCs from mouse B resulted in Axl binding and 4 out of 4 of the HC from mouse D resulted in Axl binding.

For mouse A and C supernatants from the LEE co-transfections of the single HC with the pooled LCs were analyzed for Axl binding of the produced antibody mixtures by the diversity screen. This screen enabled both the identification of Axl binding HCs and a rough epitope mapping, by identifying the loss of binding of antibodies to Axl variants. From mouse A approximately 40 % of the HCs bound to human Axl, most of which lost binding either to the Igl or FNIII-2 domain, when these domains were replaced by the mouse equivalent. From mouse C approximately 70 % of the HCs bound to human Axl, most of which lost binding either to the Igl or Ig2 domain, when these domains were replaced by the mouse equivalent. Based on binding as determined by Axl ELISA or the diversity screen, HC sequence information and loss of binding to specific Axl domains in the diversity screen a total of 12 unique HCs were selected for determination of the best LC.

Co-transfection of HCs with single LCs

Each single HC LEE of the 12 unique selected HCs was cotransfected with 96 single LC LEEs from the LC pool of the corresponding mice.

LC selection of Axl binding antibodies

Supernatants of the LEE expression of the single HC / LC combinations were analyzed for Axl binding of the produced antibody by the Axl ELISA. For each HC at least 6 LCs were found and a single LC was selected as best, based on both the ELISA results and the LC sequence information. In figure 1 an illustrative example of this LC screen is shown. Axl binding antibodies were identified from all 4 mice, even the mice which were not successful in the hybridoma process.

Binding affinity of antibody 511 The affinity of 1 anti-AXL antibody (clone 511) was determined.

Affinity was determined using Bio-Layer Interferometry on a ForteBio OctetRED384. Anti- human Fc Capture (AHC) biosensors (ForteBio, Portsmouth, UK; cat no. 18-5064) were loaded for 150 s with hlgG (1 μg/mL) aiming at a loading response of 1 nm. After a baseline (150 s) the association (1000 s) and dissociation (2000 s) of AXLECDHis (as described in Example 1) was determined, using a concentration range of 10 μg/mL - 0.16 μg/mL (218 nM - 3 nM) with 2-fold dilution steps. For calculations, the theoretical molecular mass of

AXLECDHis based on the amino acid sequence was used, i.e. 46 kDa. Experiments were carried out on an OctetRED384, while shaking at 1000 rpm and at 30°C. Each antibody was tested in three independent experiments.

Data was analyzed with ForteBio Data Analysis Software v7.0.3.1, using the 1 : 1 model and a global full fit with 1000 s association time and 1000 s dissociation time unless stated otherwise. A dissociation time of 1000 s (instead of the 2000 s dissociation time that was acquired) was used since this resulted in better fits. Data traces were corrected by subtraction of a reference curve (antibody without AXLECDHis), the Y-axis was aligned to the last 5 s of the baseline, and interstep correction as well as Savitzky-Golay filtering was applied .

The affinity (K_D) of clone 511 for Axl was 23*10^"9M (k_on 1.7*10⁵ 1/Ms and a k_dis of 3.9*10^"3 1/s) .

Exa m p ie 2

Sort il in ex p ression con st ru cts

The encoding sequences for full-length human Sortilin (SORT; UniProt Q99523),

SORTECDBAP (the extra cellular domain (ECD) of sortilin fused to a C-terminal biotin acceptor peptide (BAP)) and SORTECDHis (the ECD of sortilin fused to a C-terminal His tag)) were made synthetically and fully codon optimized (GeneArt, Regensburg, Germany) . The sequences contained suitable restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence (Kozak et al . (1999) Gene 234 : 187-208) . The sequences were cloned in the mammalian expression vector pcDNA3.3 (Invitrogen), which resulted in the vectors p33- SORT, p33-SORTECDHIS and p33-SORTECDBAP.

The sortilin ectodomain (sSortilin) that was used for immunization, encompassing the entire coding region of the N-terminal part of human sortilin (GenBank CAA66904.2) fused to a C- terminal polyhistidine tag inserted, was produced and purified as described (Andersen et al. (2010) J . Biol. Chem . 285 : 12210-12222) . Tra nsient ex pression in H EK293 F a nd CH O cells

HEK293F cells were transfected with p33-SORTECDHIS and p33-SORTECDBAP plasmid DNA, using 293fectin (Invitrogen) according to the manufacturer's instructions. p33-SORTECDBAP was co-transfected with a construct expressing the E. coli BirA enzyme, which catalyzes the biotinylation of the BAP tag in vivo. p33-SORT was transiently transfected in the Freestyle™ CHO-S (Invitrogen) cell line using Freestyle MAX transfection reagent (Invitrogen) .

Expression of full-length SORT was tested by means of FACS analysis.

SORTECDHIS was expressed in HEK293F cells and was purified by immobilized metal affinity chromatography as described supra. I m m u n izat ion of t ran sgen ic m i ce

Anti-Sortilin antibodies were derived from the following immunizations of HuMAb mice: Two HCol7, one HCo20, one HCol2-BALB/c, two HCol7-BALB/c and one HCo20-BALB/c mouse (Bristol-Myers Squibb, Redwood City, CA, USA) were immunized alternating intraperitoneally (IP) with 20 μg of sSortilin and subcutaneously (SC; at the tail base) with 20 μg of the protein with an interval of 14 days. In total, 8 immunizations were performed, 4 IP and 4 SC. The first immunization was performed in complete Freunds' adjuvant (CFA; Difco

Laboratories, Detroit, MI, USA) . All subsequent immunizations were performed in incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, MI, USA) . In addition, one HCo20, two

HCol2-BALB/c, one HCol7-BALB/c and one HCo20-BALB/c mouse were immunized according to the same schedule using Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA) . A maximum of eight immunizations were performed, 4 IP and 4 SC. When serum titers were found to be sufficient (serum dilution of 1/200 or further found positive in a homogeneous antigen-specific screening assay as described on at least two sequential, biweekly, screening events), mice were additionally boosted twice intravenously (IV) with 10 μg sSortilin protein in 100 μί PBS, four and three days before the animals were sacrificed . Spleen and lymph nodes flanking the abdominal aorta and caval vein were collected and cells were isolated, counted and stored in liquid N2. H om ogeneous an t igen-specif ic screen in g assay

Mouse sera and HEK-F antibody transfectoma supernatants were analyzed in a high throughput homogeneous screening using the ImageXpress Velos ("IsoCyte"), (Molecular Devices, Sunnyvale, CA) for the presence of anti-Sortilin antibodies. The titer screen assay used streptavidin beads coupled with biotinylated SORTECDBAP to detect human anti-Sortilin antibodies. In the screening assay, TC6003-SORT and streptavidin beads coupled to

SORTECDBAP were used to detect human anti-Sortilin antibodies. Wild type CHO-S cells were used to measure non-specific binding . Samples were added to the cells or beads to allow binding to Sortilin. Subsequently, antibody binding was detected using a fluorescent conjugate (DyLight649-conjugated AffiniPure Goat anti-Human IgG Fc fragment specific, Jackson ImmunoResearch) . Mouse anti-human Sortilin antibody (Genscript/Lundbeck, MAB 1F2F4 and MAB 3B5D4), detected with a mouse IgG specific fluorescent conjugate

(DyLight649-conjugated AffiniPure Goat anti-Mouse IgG Fc fragment specific, Jackson ImmunoResearch) was used as positive control and Human ChromPure (Jackson

ImmunoResearch) detected using a fluorescent conjugate (DyLight649-conjugated AffiniPure Goat anti-Human IgG Fc fragment specific, Jackson ImmunoResearch) was used as a negative control . The screening assay reports the number of objects (NO) with fluorescence intensities above threshold and the total fluorescence intensity (TI) for all objects per well .

I solat ion of RNA from sp lee n cel ls Total RNA was isolated from spleen cells using the Mini RNA easy kit (Qiagen) . First strand cDNA for 5'-RACE was synthesized using 150 ng of RNA using the SMART RACE cDNA Amplification kit (Clonetech), PrimeScript Reverse Transcriptase (TAKARA BIO) and the SMART IIA oligo and oligodT as primers. VL encoding regions were amplified by PCR using Advantage 2 polymerase (Clontech), the primers RACEkLIC4shortFW2 (320 nM),

RACEkLIC4LongFW2 (80 nM) and RACEkLICRV_PmlA3 (400 nM), performing 35 cycles of 30 seconds at 95°C, and 1 minute at 68°C. VH encoding regions were amplified by PCR using Pfu Ultra II Fusion HS DNA polymerase (Stratagene), the primers RACEG 1 LIC3shortFW (320 nM), RACEG lLIC3longFW (80 nM) and RACEG 1LIC3RV2 (400 nM), performing 40 cycles of 20 seconds at 95 °C, 20 seconds at 66°C and 30 seconds at 72°C, ending with a finale extension step of 3 minutes at 72°C. VH or VL encoding PCR products were separated using agarose gel electrophoresis and DNA products of the correct size were cut from the gel and purified using the Qiagen MiniElute kit. VH and VL coding regions amplified by PCR were cloned, in frame, in the mammalian expression vectors pGlf (containing the human IgGl constant region encoding DNA sequence) for the VH region and pKappa (containing the kappa light chain constant region encoding DNA sequence) for the VL region, by ligation independent cloning (Aslanidis, C. and P. J . de Jong, Nucleic Acids Res 1990; 18(20) : 6069- 74) in E.coli strain DH5aTlR (DH5) (Life technologies), yielding single bacterial colonies each containing a single HC or LC expression vector

Generat ion of H C an d LC LEE p rod u cts, seq u en ce p lates a nd t ransfect io n sam p les.

E.coli DH5 colonies transformed with plasmids encoding HC or LC proteins were picked to a 384 well PCR plate using the BiOcto-Pik K6-2 colonypicker (KBiosystems, UK). Each colony was transferred into a single well with 30 μΙ LEE PCR buffer containing lx AccuPrime PCR buffer, 2 μ Μ CMV P f(RRA2), 2 μ Μ Tk pA r (RRA2) primer (Sigma-Aldrich, US) and 0.6 U AccuPrime polymerase (Invitrogen, US) . After picking, each 384 well plate contained 176 HC and 176 LC constructs from a single mouse (first 2 columns are kept empty for process related controls) . Typically 12 mice were included per project resulting in twelve 384 well plates per project (2112 HC PCR products and 2112 LC PCR products) . After picking, the plates were sealed and the expression cassettes were amplified by PCR (15' 94 °C, [30" 94 °C, 30" 55 °C, 5' 68 °C] 35x, 4 °C) .

The source plates containing LEE PCR product were used to generate several daughter plates. From a single 384 well plate, one backup plate (5 μΙ PCR product per well), one sequence plate (5 μΙ PCR product in 15 μΙ PCR grade water (B.Braun) per well) and two transfection plates were generated. The sequence plates are used to sequence the PCR products using Sanger sequencing using the pCEP4-for sequence primer (Beckman Coulter Genomics, UK).

Gene rat ion of H C + LC pool t ra nsfect ion p lates

Per mouse, the LCs were divided and pooled into two LC pools of 88 LC each. One LC pool contained 880 μΙ LC LEE PCR product (10 μΙ LEE PCR product per LC) and PCR grade water was added to compensate for dead volumes. The transfection plates contains 1 μΙ PCR grade water, 2 μΙ HC and 2 μΙ LC pool (containing 88 LCs per pool) per well. Each HC is transfected twice, once with LC pool 1 and once with LC pool 2 (176 HCs x 2 LC pools = 352

transfections per plate x 12 plates = 4224 transfection reactions). The second transfection plate was kept as backup. The plates were stored at -20 °C before being process further.

Seq uen ce a na lysis

The sequence traces (abl format) obtained for the twelve 384 well plates were analyzed. The analysis determines the closest mouse or human germline gene using MAFFT-distance (http://mafft.cbrc.ip/aliqnment/software/^') against a human germline reference database. Subsequently, the framework and complementarity determining regions were determined by aligning the query sequence against a prealigned and annotated germline reference database using MAFFT (http://mafft.cbrc.ip/aliqnment/software/). The germline reference sequences were aligned according to the IMGT numbering scheme via the -add option, BLOSUM62 distance matrix and a default gap opening penalty of 1.53. Finally, sequences with similar CDR3 were clustered together using CDHIT ( http://weizhonq-lab.ucsd.edu/cd-hit/). For

CDHIT the following parameters were used: -c 0.8 -I 6 -d 50 -s 0.6 -g 1 -G 0 -aS 0.95. For the LC library, typically 80-100% of the sequences were identified as VL containing sequences. For the HC library, the frequency of identifying VH sequences is slightly lower and typically 60-100% of the sequences are identified as VH containing sequences.

Mouse Identified HC sequence Identified LC sequence

544376 69% 96%

552971 68% 91%

552962 61% 92%

544366 95% 97%

554572 90% 95% 545922 86% 84%

555553 88% 93%

545928 95% 97%

544362 89% 96%

552936 97% 94%

544378 91% 90%

552934 79% 97%

Table 2: Success rate of identifying HC or LC variable domains in sequences obtain from LEE PCR products. Per mouse, a maximum number of 176 HC and 176 LC are expected. The percentage of identified HC or LC sequences is reported . Tra nsfect ion of H C + LC pools

Per well 0.24 μΙ 293Fectin (Invitrogen, US ) and 4.76 μΙ Opti-MEM (Gibco, US) were mixed and incubated for 5 minutes at room temperature. Next, the Fectin/Opti-MEM mix was added to the 5 μΙ DNA and incubated for 30 minutes at room temperature. Finally, 8.3 μΙ of the Fectin/Opti-MEM/DNA mix was added to 117.5 μΙ HEK29F cells. During all procedures, the plates with HEK293F cells were shaken to keep the cells in suspension. After transfection, cells are incubated at 37 °C/8% C02 for 5 days.

Five days post transfection, the supernatant was harvested .

Screen i n g of H C+ LC pool com b i nat ions .

CHO cells expressing Sortilin (TC6003-SORT) or beads coupled with the extracellular domain of Sortilin (SORTECDBAP) were used to screen for binding of the HC: LC pool combinations in a homogeneous binding as described supra.

As mentioned above the screening assay reports the number of objects (NO) with fluorescence intensities above threshold and the total fluorescence intensity (TI) for all objects per well . The complexity of the dataset was reduced by grouping samples belonging to the same HC CDR3 cluster together. The average total intensity signal and average number of objects was compared between clusters to identify those HC CDR3 clusters that yielded the strongest binding. The number of HC CDR3 clusters that contained samples identified as binder ranged from 1 to 9 per mouse. A typical example of the number of binding samples per HC CDR3 cluster and the average binding signal per HC CDR3 cluster for one mouse is shown in FIGURE 2. Select ion of m ax 96 H C a nd correspon d i n g LC pools

Out of the 4224 samples, 986 samples were identified with binding levels above threshold (number of objects > 50). Out of the binders, 96 HCs were selected where for each HC cluster with binders at least 1 HC with the LC pool that resulted in the strongest binding was selected. When sequence liabilities were identified , alternative clones were included. For some HC CDR3 clusters multiple samples were selected that differed in their HC CDR3 sequence or showed variations in the variable domain. The selected samples were derived from 38 different HC CDR3 clusters.

Generat ion of H C w it h si n g le LC t ransfect ion m ixt u res

In the second screening round, the 96 selected HCs were tested with all individual LCs of the corresponding selected LC pools (96 x 88 LCs) . Since the remainder of the 30 μΙ HC LEE PCR product was not sufficient to test again with 88 LCs (88 transfections) the selected HCs were hitpicked and reamplified using (nested) PCR in 2x 30 μΙ LEE PCR buffer. The 60 μΙ HC LEE PCR product was pooled and used to generate HC: LC mixtures in a 1 : 1 ratio. The HC LEE PCR product was diluted 13.7 times and 8.2 μΙ was mixed with 1.8 μΙ 3 times diluted LC LEE product. Of this transfection mix, 5 μΙ was used for transfection and 5 μΙ was kept as backup.

The HC: LC combinations were transfected, expressed and supernatant was harvested as described supra.

Screen i n g of H C + LC com bi nat ion s

CHO cells expressing Sortilin (TC6003-SORT) or beads coupled with the extracellular domain of Sortilin (SORTECDBAP) were used to screen for binding of the HC: LC pool combinations in a homogeneous binding as described supra.

Per HC the LCs that resulted in the strongest binding signals were selected for a dose response screen to determine the affinity. Out of 8448 samples, 542 samples were selected for the dose response screen. The selected HC+LC combination were derived from 11 mice. An example for the number of binding samples per LC CDR3 cluster and the average binding signal per LC CDR3 cluster for a single is shown in FIGURE 3.

D ose respon se scree n for selected H C/ LC com bi nat ions

Antibody concentration in supernatant was measured by BioLayer Interferometry using the Octet RED (ForteBio, US). For the dose response assay, antibody serial dilutions series were generated starting from a maximum concentration of 1 ug/ml. These dilution series were used in a homogeneous binding assay described in supra. Per HC the LC yielding the lowest EC50 value was selected. Out of the 542 samples selected for dose response screen, 74 samples were selected based on the EC50 values. These antibodies were derived from 11 mice. Table 3 shows an example for the dose response data obtained for a single HC with alternative LCs. Table 4 shows EC50 and max binding data for a single HC with alternative LCs indicating that LC choice can affect affinity.

Table 3. Dose response data obtained for a single HC with alternative LCs. Affinity data using Octet.

Ab code _D (M)

IgGl-6003-5008 9.5*1(Γ⁰⁹

IgGl-6003-5009 3.6*1(Γ⁰⁹

IgGl-6003-5011 3.5*1(Γ⁰⁹

IgGl-6003-5027 5.7*1(Γ⁰⁹

IgGl-6003-5028 1.2*1CT⁰⁸

IgGl-6003-5029 8.9*1(Γ⁰⁹

IgGl-6003-5034 1.1*1CT⁰⁸

IgGl-6003-5035 3.3*1(Γ⁰⁸

IgGl-6003-5036 3.6*1(Γ⁰⁸

IgGl-6003-5041 3.2*1(Γ⁰⁸

IgGl-6003-5042 7.8*1(Γ⁰⁸

Table 4. LC dependent affinity modulation. The same HC was tested with different LCs in a dose response assay. For different LCs, different EC50 and max binding values were obtained.

Mouse SampleJD EC50 (ug/ml) Max binding (A.U.)

(TC6003-SORT (mut)) (TC6003-SORT (mut))

544362 R1D03-P1C20 0.0209 1.00*10^+os

544362 R1D03-P1C06 0.0269 5.71*10⁺⁰⁷

544362 R1D03-P1O08 0.0270 1.13*10^+os

544362 R1D03-P1I12 0.0291 1.36*10^+os

544362 R1D03-P1A16 0.0360 1.18*10^+0S

544362 R1D03-P1M18 0.0398 8.96*10⁺⁰⁷

544362 R1D03-P1K18 0.0412 1.08*10^+0S

544362 R1D03-P1M22 0.0417 1.09*10^+0S

544362 R1D03-P1A20 0.0421 8.94*10⁺⁰⁷

544362 R1D03-P1O20 0.0446 9.78*10⁺⁰⁷

544362 R1D03-P1K20 0.0454 7.89*10⁺⁰⁷

544362 R1D03-P1K24 0.0591 4.09*10⁺⁰⁷

544362 R1D03-P1K04 0.0663 1.07*10^+0S 544362 R1D03-P1K12 0.0682 1.84*10^+0S

544362 R1D03-P1A24 0.0842 9.39*10⁺⁰⁷

544362 R1D03-P1E04 0.0846 5.38*10⁺⁰⁷

Plasm id rescue

The plasmids from the lead panel encoding HC and LC sequences were rescued from the LEE PCR mix. LEE PCR was diluted lOOx using PCR grade water (B.Braun) and 1 μΙ was transformed to One Shot® MAX Efficiency® DH5a™-TlR Competent Cells (Invitrogen, US). Colonies were picked by hand, propagated and plasmid DNA was isolated. The obtained HC and LC sequence were confirmed by Sanger sequencing.

Out of the 74 lead candidates, a panel of 48 antibodies was randomly selected. For each antibody the HC and LC plasmid sequence was rescued and maxi prepped. Affinity analysis:

Purified antibodies were diluted in PBS to a concentration of 2 μg/ml. The antibody dilutions were immobilized on protein A sensors by incubating the antibodies with protein A sensors (ForteBio) for 200 seconds. After a kinetic run, sensors were discarded and antibodies were captured to fresh protein A sensors. The captured antibodies were incubated with

SORTECDHis analyte spanning a dilution range of 1.25μg/ml-0.156μg/ml (16 nM - 2 nM). After association of the analyte with the antibodies, the tips were incubated in PBS allowing the analyte to dissociate. The association time was 1000 seconds while the dissociation time was kept at 1500 seconds. The observed binding curves were fitted with a one to one interaction model (ForteBio) . Antibodies with affinities for SORTECDHis were found in the nM range (Table 5).

Table 5. Dose response curves for the 48 HC/LC combinations that where selected as lead panel

Ab code EC50 (Mg/ml) Max binding (A.U.)

(TC6003-SORT (mut)) (TC6003-SORT (mut))

IgGl-6003 -5001 1 09*10^~02 5 96*10⁺⁰⁷

IgGl-6003 -5002 6 57*10^~03 6 33*10⁺⁰⁷

IgGl-6003 -5003 7 82*10^~03 6 10*10⁺⁰⁷

IgGl-6003 -5004 1 81*10^~02 2 34*10⁺⁰⁷

IgGl-6003 -5005 0 00*10⁺⁰° 0 00*10⁺⁰°

IgGl-6003 -5006 1 12*10^~02 7 63*10⁺⁰⁷

IgGl-6003 -5007 1 29*10^~02 5 51*10⁺⁰⁷ IgGl-6003-5008 7.94*1(Γ⁰³ 5.68*10⁺⁰⁷

IgGl-6003-5009 7.20*1(Γ⁰³ 6.97*10⁺⁰⁷ IgGl-6003-5010 ND ND

IgGl-6003-5011 7.76*1(Γ⁰³ 5.90*10⁺⁰⁷ IgGl-6003-5012 8.77*1(Γ⁰³ 2.94*10⁺⁰⁷

IgGl-6003-5013 3.67*1(Γ⁰² 3.42*10⁺⁰⁷ IgGl-6003-5014 1.27*10^~°² 6.61*10⁺⁰⁷

IgGl-6003-5015 1.69*10^~°² 5.74*10⁺⁰⁷ IgGl-6003-5016 1.05*10^~°² 7.93*10⁺⁰⁷

IgGl-6003-5017 ND ND IgGl-6003-5018 1.59*10⁺⁰⁰ 6.49*10⁺⁰⁷

IgGl-6003-5019 5.37*1(Γ⁰³ 5.97*10⁺⁰⁷ IgGl-6003-5020 6.59*1(Γ⁰³ 5.97*10⁺⁰⁷

IgGl-6003-5021 1.06*10^~°² 7.18*10⁺⁰⁷ IgGl-6003-5022 7.42*1(Γ⁰³ 6.79*10⁺⁰⁷

IgGl-6003-5023 1.24*1(Γ⁰² 7.68*10⁺⁰⁷ IgGl-6003-5024 1.22*10^~°² 6.62*10⁺⁰⁷

IgGl-6003-5025 2.25*1(Γ⁰² 5.96*10⁺⁰⁷ IgGl-6003-5026 4.98*1(Γ⁰³ 7.05*10⁺⁰⁷

IgGl-6003-5027 5.12*1(Γ⁰³ 5.19*10⁺⁰⁷ IgGl-6003-5028 5.48*1CT⁰³ 4.52*10⁺⁰⁷

IgGl-6003-5029 8.44*1CT⁰³ 8.05*10⁺⁰⁷ IgGl-6003-5030 1.03*10^~°² 6.23*10⁺⁰⁷

IgGl-6003-5031 5.56*1(Γ⁰³ 4.71*10⁺⁰⁷ IgGl-6003-5032 7.94*1(Γ⁰³ 7.38*10⁺⁰⁷

IgGl-6003-5033 1.06*1(Γ⁰² 6.10*10⁺⁰⁷ IgGl-6003-5034 7.75*1(Γ⁰³ 5.55*10⁺⁰⁷

IgGl-6003-5035 7.41*1(Γ⁰³ 6.06*10⁺⁰⁷ IgGl-6003-5036 7.13*1(Γ⁰³ 5.30*10⁺⁰⁷

IgGl-6003-5037 1.18*1CT°² 5.48*10⁺⁰⁷ IgGl-6003-5038 1.24*1(Γ⁰² 4.22*10⁺⁰⁷

IgGl-6003-5039 5.49*1CT⁰³ 7.18*10⁺⁰⁷ IgGl-6003-5040 8.47*1(Γ⁰³ 4.33*10⁺⁰⁷

IgGl-6003-5041 4.86*1(Γ⁰³ 7.89*10⁺⁰⁷ IgGl-6003-5042 4.98*1(Γ⁰³ 6.04*10⁺⁰⁷

IgGl-6003-5043 6.11*1(Γ⁰³ 3.78*10⁺⁰⁷ IgGl-6003-5044 7.86*1(Γ⁰³ 7.20*10⁺⁰⁷

IgGl-6003-5045 5.55*1(Γ⁰² 2.56*10⁺⁰⁷ IgGl-6003-5046 1.19*10^~°² 6.00*10⁺⁰⁷

IgGl-6003-5047 ND ND IgGl-6003-5048 1.40*1CT⁰² 1.98*10⁺⁰⁷

Claims

Claims

1. A method for the selection of an individual antibody, the antibody binding at least one antigen(s) of interest and comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones

expressing HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Cotransfecting cells with each one individual clone expressing a specific HC of said HC pool together with said LC pool comprising a plurality of individual clones expressing LC to express antibodies from said cells consisting of said specific HC randomly combined with LC(s) from said LC pool, this co-transfection being performed for each individual clone of said HC pool; iii) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual

HC clones; iv) Cotransfecting cells with the clone(s) expressing each HC selected under step iii) together with each one individual clone expressing a specific LC of said LC pool to express antibodies with combined HC and LC; and v) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said HC selected under step iii) to provide the best combined LC and HC antibody.

2. A method for the selection of an individual antibody, the antibody binding an antigen(s) of interest and comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones

expressing HC and a first LC pool comprising a plurality of individual clones expressing LC; ii) Cotransfecting cells with each one individual clone expressing a specific LC of said LC pool together with said HC pool comprising a plurality of individual clones expressing HC to express antibodies from said cells consisting of said specific LC randomly combined with HC(s) from said HC pool, this co-transfection being performed for each individual clone of said LC pool; iii) Screening for and selecting one or more LCs based on pre-set criteria of said antibodies obtained under step ii) to provide one or more individual LC clones; iv) Cotransfecting cells with the clones expressing each LC selected under step iii) together with each one individual clone expressing a specific HC of said HC pool to express antibodies with combined LC and HC; and v) Screening for and selecting one or more HCs based on pre-set criteria of said antibodies obtained under step iv) when in combination with said LC selected under step iii) to provide the best combined LC and HC antibody.

3. A method according to claims 1 or 2, which method comprises the preceding steps of a) Providing a plurality of individual clones expressing HC in one or more first HC pools and a plurality of individual clones expressing LC in one or more first LC pools; b) Cotransfecting cells with each of said first HC pool together with each of said first LC pool to express antibodies from said cells consisting of randomly combined HC and LC(s) from said first HC and LC pools, this co-transfection being performed for each individual first HC pool and/or for each individual first LC pool; c) Screening for and selecting the HC pools and/or the LC pools based on pre-set criteria of said antibodies obtained under step b) to provide one or more second HC pools and/or one or more second LC pools; d) Using the pools obtained under step c) in step i) of claims 1 or 2.

4. The method according to claim 3, wherein steps a)-c) are repeated one or more times for the selected HC pools and/or LC pools so that said second pools selected under step c) becomes said first LC pools and/or HC pools of step a) before proceeding with step d) .

5. The method according to any of claims 1-4, which method comprises the preceding steps of a) isolating RNA comprising RNA encoding the heavy chain of antibodies from each of a plurality of antibody expressing cells derived from a plurality of antibody producing individuals such as naive individuals or individuals immunized with said same antigen of interest; b) preparing cDNA of said RNA encoding HC of said antibodies and cloning the cDNA into an expression system; c) selecting individual clones from step b) to prepare a plurality of individual clones expressing HC (HC pool); and d) using the pools obtained under step c) in step i) of claims 1-2 or in step a) of claims 3-4.

6. The method according to any one of claims 1-5, which method comprises the preceding steps of a) isolating RNA comprising RNA encoding the light chain of antibodies from each of a plurality of antibody expressing cells derived from a plurality of antibody producing individuals, such as naive or individuals immunized with said same antigen of interest; b) preparing cDNA of said RNA encoding LC of said antibodies and cloning the cDNA into an expression system; and c) selecting individual clones from step b) to prepare a plurality of individual clones expressing LC (LC pool); and d) using the pools obtained under step c) in step i) of claims 1-2 or in step a) of claims 3-4.

7. The method according to any one of claims 5-6, wherein said plurality of antibody expressing cells are B lymphocytes or spleen cells obtained directly from said individual.

8. The method according to any one of claims 1-2, wherein steps i)-iii) and optionally steps iv)-v) are repeated under different pre-set criteria for the selected HCs so that the one or more individual HC clones provided under step iii) and/or step v) becomes the LC pool and/or HC pool of step i) .

9. The method according to any one of the claims 3-4 wherein steps a)-c) are repeated under different pre-set criteria for the selected HCs and/or LCs, so that the one or more individual HC or LC clones provided under step c) becomes the first HC and/or LC pool of step a).

10. The method according to any one of claims 1-9, wherein said HC(s) and said LC(s) bind the same antigen.

11. The method according to any one of claims 1-9 wherein said HC(s) and said LC(s) bind different antigens.

12. The method according to any one of claims 1-11, wherein said plurality of individual clones expressing HC (HC pool) and/or said plurality of individual clones expressing LC (LC pool) is obtained from a synthetic library, genomic DNA, protein sequence information, a cDNA library, T cell receptor library, or from the method steps described in claims 3-4.

13. The method according to any one of claims 1-12, wherein said plurality of individual clones expressing HC (HC pool) and/or said plurality of individual clones expressing LC (LC pool) derives from antibody producing cells, such as cells of the blood circulation, bone marrow cells, spleen cells, such as splenocytes of an animal, such as an immunized animal or naive animal, such as mouse, rabbit, rat, guinea pig, or camel.

14. The method according to any one of claims 1-13, which method comprises a step of preparation of Linear expression elements (LEE's) encoding said HC and/or LC.

15. The method according to any one of claims 1-14, wherein said first and/or said second LC pool consist of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing LC.

16. The method according to any one of claims 1-15, wherein said first and/or said second HC pool consist of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1000000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 individual clones expressing HC.

17. The method according to any one of claims 1-16, wherein said first and/or second HC pool and/or said first and/or second LC pool derives from at least 2, 3, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 2-100, 10-100, 20-100, 30-100, 40-100, 50-100, or 60-100 individual animals, such as mouse, rabbit, rat, guinea pig, or camel, naive or immunized with the same or different specific antigen.

18. The method according to any one of claims 5-17, wherein primers specific for the amplification of IgM or IgGl HC VHs are used under step b) in the amplification of HC encoding cDNA molecules.

19. The method according to any one of claims 5-18, wherein primers specific for the amplification of kappa or lambda LCs are used under step b) in the amplification of LC encoding cDNA molecules.

20. The method according to any one of claims 1, 3-19, wherein the pre-set criteria for screening and selecting the HCs under step iii) are based on specific binding, such as in a qualitative binding assay determining binding to a specific antigen.

21. The method according to any one of claims 2-20, wherein the pre-set criteria for screening and selecting the LCs under step iii) are based on specific binding, such as in a qualitative binding assay determining binding to a specific antigen.

22. The method according to any preceding claim, wherein said first and/or second HC pool and/or said first and/or second LC pool derives from a transgenic animal, such as mouse, rabbit, rat, camelid immunized to produce fully human or humanized antibodies or from sequence information of fully human or humanized antibodies.

23. The method according to any one of claims 5-22, wherein under step b) VH and VL coding regions are amplified by 5' RACE or PCR before cloning into an expression system.

24. The method according to any one of claims 5-23, wherein under step b) VH and VL coding regions are cloned into a bacterial, yeast, plant, or mammalian expression vector.

25. The method according to any preceding claim, wherein the pre-set criteria for screening and selecting the combined HC and LC antibody or antibodies in step v) is based on antigen binding ELISA, or homogeneous binding assay.

26. The method according to any preceding claim, which method screens for and selects antibodies with a dissociation constant (K_D) lower than 10 ^M, such as lower than 5x10 ⁷M,

-8 -9 -9 such as lower than 10 M, such as lower than 5x10 M, , such as lower than 10 M, such as lower than 5x10 ¹⁰M.

27. The method according to any preceding claim, wherein the pre-set criteria for screening and selecting of the combined HC and LC antibody or antibodies in step v) is based on a functional assay screen such as but not limited to internalization assay selecting antibodies inducing target internalization or lysosomal targeting or target down modulation, or signalling inhibition/activation or cell kill or target (cells) crosslinking.

28. A method for the production of a recombinant antibody binding an antigen or antigens of interest and comprising a heavy chain (HC) and a light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing HC and a first LC pool comprising a plurality of individual clones expressing LC;

ii) Cotransfecting cells with each one specific clone expressing a specific HC of said HC pool together with a composition comprising all clones of said LC pool to express antibodies from said cells consisting of said specific HC randomly combined with an LC from said LC pool, this co-transfection being performed for each individual clone of said HC pool;

iii) Screening for and selecting the best HC of said antibodies obtained under step ii) to provide individual HC clones;

iv) Cotransfecting cells with clones expressing each HC selected under step iii) together with each one specific clone expressing a specific LC of said LC pool to express antibodies with combined HC and LC; v) Screening for and selecting the best LC of said antibodies obtained under step iv) when in combination with said HC selected under step iii) to provide the best combined LC and HC antibody; and

vi) Inserting the nucleic acids encoding the said selected antibody from v) in an antibody producing recombinant host cell, expressing the antibody, isolating and purifying the antibody.

29. A method for the production of a recombinant antibody binding an antigen or antigens of interest and comprising a heavy chain (HC) and a light chain (LC), which method comprises the steps of i) Providing a first HC pool comprising a plurality of individual clones expressing HC and a first LC pool comprising a plurality of individual clones expressing LC;

ii) Cotransfecting cells with each one specific clone expressing a specific LC of said LC pool together with a composition comprising all clones of said HC pool to express antibodies from said cells consisting of said specific LC randomly combined with an HC from said HC pool, this co-transfection being performed for each individual clone of said LC pool;

iii) Screening for and selecting the best LC of said antibodies obtained under step ii) to provide individual LC clones;

iv) Cotransfecting cells with clones expressing each LC selected under step iii) together with each one specific clone expressing a specific HC of said HC pool to express antibodies with combined LC and HC;

v) Screening for and selecting the best HC of said antibodies obtained under step iv) when in combination with said LC selected under step iii) to provide the best combined HC and LC antibody; and

vi) Inserting the nucleic acids encoding the said selected antibody from v) in an antibody producing recombinant host cell, expressing the antibody, isolating and purifying the antibody.

30. The methods according to any of claims 28 or 29, which method comprises the step as described in any one of claims 3-27.

31. Antibody obtainable by or obtained by the method according to any of claims 28-30.

32. Antibody according to claim 31 for use in a method of treating a disease or disorder.