AU764097B2 - Protein/(poly)peptide libraries - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): MORPHOSYS GESELLSCHAFT FUR PROTEINOPTIMIERUNG MBH Invention Title: PROTEIN/(POLY)PEPTIDE LIBRARIES The following statement is a full description of this invention, including the best method of performing it known to me/us: WO 97/08320 PCT/EP96/03647 Protein/(Poly)peptide Libraries Field of the Invention The present invention relates to synthetic DNA sequences which encode one or more collections of homologous proteins/(poly)peptides, and methods for generating and applying libraries of these DNA sequences. In particular, the invention relates to the preparation of a library of human-derived antibody genes by the use of synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome. Furthermore, the invention relates to the use of a single consensus antibody gene as a universal framework for highly diverse antibody libraries.

Background to the Invention All current recombinant methods which use libraries of proteins/(poly)peptides, e.g.

antibodies, to screen for members with desired properties, e.g. binding a given ligand, do not provide the possibility to improve the desired properties of the members in an easy and rapid manner. Usually a library is created either by *inserting a random oligonucleotide sequence into one or more DNA sequences cloned from an organism, or a family of DNA sequences is cloned and used as the library. The library is then screened, e.g. using phage display, for members which show the desired property. The sequences of one or more of these resulting molecules are then determined. There is no general procedure available to improve these molecules further on.

Winter (EP 0 368 684 B1) has provided a method for amplifying (by PCR), cloning, and expressing antibody variable region genes. Starting with these genes he was able to create libraries of functional antibody fragments by randomizing the CDR3 of the heavy and/or the light chain. This process is functionally equivalent to the natural process of VJ and VDJ recombination which occurs during the development of Bcells in the immune system.

However the Winter invention does not provide a method for optimizing the binding affinities of antibody fragments further on, a process which would be functionally equivalent to the naturally occurring phenomenon of "affinity maturation", which is provided by the present invention. Furthermore, the Winter invention does not provide for artificial variable region genes, which represent a whole family of -1A SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 structurally similar natural genes, and which can be assembled from synthetic DNA oligonucleotides. Additionally, Winter does not enable the combinatorial assembly of portions of antibody variable regions, a feature which is provided by the present invention. Furthermore, this approach has the disadvantage that the genes of all antibodies obtained in the screening procedure have to be completely sequenced, since, except for the PCR priming regions, no additional sequence information about the library members is available. This is time and labor intensive and potentially leads to sequencing errors.

The teaching of Winter as well as other approaches have tried to create large antibody libraries having high diversity in the complementarity determining regions (CDRs) as well as in the frameworks to be able to find antibodies against as many different antigens as possible. It has been suggested that a single universal framework may be useful to build antibody libraries, but no approach has yet been successful.

Another problem lies in the production of reagents derived from antibodies. Small antibody fragments show exciting promise for use as therapeutic agents, diagnostic reagents, and for biochemical research. Thus, they are needed in large amounts, and the expression of antibody fragments, e.g. Fv, single-chain Fv (scFv), or Fab in the periplasm of E. coli (Skerra Plckthun, 1988; Better et al., 1988) is now used routinely in many laboratories. Expression yields vary widely, however. While some fragments yield up to several mg of functional, soluble protein per liter and OD of culture broth in shake flask culture (Carter et al., 1992, Plickthun et al. 1996), other fragments may almost exclusively lead to insoluble material, often found in so-called inclusion bodies. Functional protein may be obtained from thelatter in modest yields by a laborious and time-consuming refolding process. The factors influencing antibody expression levels are still only poorly understood. Folding efficiency and stability of the antibody fragments, protease lability and toxicity of the expressed proteins to the host cells often severely limit actual production levels, and several attempts have been tried to increase expression yields. For example, Knappik Plickthun (1995) could show that expression yield depends on the antibody sequence. They identified key residues in the antibody framework which influence expression yields dramatically. Similarly, Ullrich et al. (1995) found that point mutations in the CDRs can increase the yields in periplasmic antibody fragment expression. Nevertheless, these strategies are only applicable to a few antibodies.

Since the Winter invention uses existing repertoires of antibodies, no influence on expressibility of the genes is possible.

-2- SUBSTITUTE SHEET (RULE 26) I- Pae~r),, were not lodged with this application WO 97108320 PCT/EP96/03647 sequences at the right position without kncwir.g !h2 actual sequence or having to determine the sequence of the individual library member.

Additionally, new information about amino acid residues important for binding, stability, or solubility and expression could be integrated into the library design by replacing existing modules with modules modified according to the new observations.

The limited number of consensus sequences used for creating the library allows to speed up the identification of binding antibodies after screening. After having identified the underlying consensus gene sequence, which could be done by sequencing or by using fingerprint restriction sites, just those part(s) comprising the random sequence(s) have to be determined. This reduces the probability of sequencing errors and of false-positive results.

The above mentioned cleavage sites can be used only if they are unique in the vector system where the artificial genes have been inserted. As a result, the vector has to be modified to contain none of these cleavage sites. The construction of a vector consisting of basic elements like resistance gene and origin of replication, where cleavage sites have been removed, is of general interest for many cloning attempts. Additionally, these vector(s) could be part of a kit comprising the above mentioned artificial genes and pre-built libraries.

The collection of artificial genes can be used for a rapid humanization procedure of non-human antibodies, preferably .of rodent antibodies. First, the amino acid sequence of the non-human, preferably rodent antibody is compared with the amino acid sequences encoded by the collection of artificial genes to determine the most homologous light and heavy framework regions. These genes are then used for insertion of the genetic sub-elements encoding the CDRs of the non-human, *preferably rodent antibody.

Surprisingly, it has been found that with a combination of only one consensus sequence for each of the light and heavy chains of a scFv fragment an antibody repertoire could be created yielding antibodies against virtually every antigen.

Therefore, one aspect of the present invention is the use of a single consensus sequence as a universal framework for the creation of useful (poly)peptide libraries and antibody consensus sequences useful therefor.

-4- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Detailed Description of the Invention The present invention enables the creation of useful libraries of (poly)peptides. In a first embodiment, the invention provides for a method of setting up nucleic acid sequences suitable for the creation of said libraries. In a first step, a collection of at least three homologous proteins is identified and then analyzed. Therefore, a database of the protein sequences is established where the protein sequences are aligned to each other. The database is used to define subgroups of protein sequences which show a high degree of similarity in both the sequence and, if information is available, in the structural arrangement. For each of the subgroups a (poly)peptide sequence comprising at least one consensus sequence is deduced which represents the members of this subgroup; the complete collection of (poly)peptide sequences represent therefore the complete structural repertoire of the collection of homologous proteins. These artificial (poly)peptide sequences are then analyzed, if possible, according to their structural properties to identify unfavorable interactions between amino acids within said (poly)peptide sequences or between said or other (poly)peptide sequences, for example, in multimeric proteins. Such interactions are then removed by changing the consensus sequence accordingly. The (poly)peptide sequences are then analyzed to identify subelements such as domains, loops, helices or CDRs. The amino acid sequence is backtranslated into a corresponding coding nucleic acid sequence which is adapted to the codon usage of the host planned for expressing said nucleic acid sequences.

A set of cleavage sites is set up in a way that each of the sub-sequences encoding the sub-elements identified as described above, is flanked by two sites which do not occur a second time within the nucleic acid sequence. This can be achieved by either identifying a cleavage site already flanking a sub-sequence of by changing one or more nucleotides to create the cleavage site, and by removing that site from the remaining part of the gene. The cleavage sites should be common to all corresponding sub-elements or sub-sequences, thus creating a fully modular arrangement of the sub-sequences in the nucleic acid sequence and of the subelements in the corresponding (poly)peptide.

In a further embodiment, the invention provides for a method which sets up two or more sets of (poly)peptides, where for each set the method as described above is performed, and where the cleavage sites are not only unique within each set but also between any two sets. This method can be applied for the creation of (poly)peptide libraries comprising for example two a-helical domains from two different proteins, where said library is screened for novel hetero-association domains.

S-TUT SHEET (RULE 6 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 In yet a further embodiment, at least two of the sets as described above, are derived from the same collection of proteins or at least a part of it. This describes libraries comprising for example, but not limited to, two domains from antibodies such as VH and VL, or two extracellular loops of transmembrane receptors.

In another embodiment, the nucleic acid sequences set up as described above, are synthesized. This can be achieved by any one of several methods well known to the practitioner skilled in the art, for example, by total gene synthesis or by PCR-based approaches.

In one embodiment, the nucleic acid sequences are cloned into a vector. The vector could be a sequencing vector, an expression vector or a display phage display) vector, which are well known to those skilled in the art. Any vector could comprise one nucleic acid sequence, or two or more nucleic sequences, either in different or the same operon. In the last case, they could either be cloned separately or as contiguous sequences.

In one embodiment, the removal of unfavorable interactions as described above, leads to enhanced expression of the modified (poly)peptides.

In a preferred embodiment, one or more sub-sequences of the nucleic acid sequences are replaced by different sequences. This can be achieved by excising the sub-sequences using the conditions suitable for cleaving the cleavage sites adjacent to or at the end of the sub-sequence, for example, by using a restriction enzyme at the corresponding restriction site under the conditions well known to those skilled in the art, and replacing the sub-sequence by a different sequence compatible with the cleaved nucleic acid sequence. In a further preferred embodiment, the different sequences replacing the initial sub-sequence(s) are genomic or rearranged genomic sequences, for example in grafting CDRs from nonhuman antibodies onto consensus antibody sequences for rapid humanization of non-human antibodies. In the most preferred embodiment, the different sequences are random sequences, thus replacing the sub-sequence by a collection of sequences to introduce variability and to create a library. The random sequences can be assembled in various ways, for example by using a mixture of mononucleotides or preferably a mixture of trinucleotides (Virnekas et al., 1994) during automated oligonucleotide synthesis, by error-prone PCR or by other methods well known to the practitioner in the art. The random sequences may be completely randomized or biased towards or against certain codons according to -6- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 the amino acid distribution at certain positions3 in know. protein seqences.

Additionally, the collection of random sub-sequences may comprise different numbers of codons, giving rise to a collection of sub-elements having different lengths.

In another embodiment, the invention provides for the expression of the nucleic acid sequences from a suitable vector and under suitable conditions well known to those skilled in the art.

In a further preferred embodiment, the (poly)peptides expressed from said nucleic acid sequences are screened and, optionally, optimized. Screening may be performed by using one of the methods well known to the practitioner in the art, such as phage-display, selectively infective phage, polysome technology to screen for binding, assay systems for enzymatic activity or protein stability. (Poly)peptides having the desired property can be identified by sequencing of the corresponding nucleic acid sequence or by amino acid sequencing or mass spectrometry. In the case of subsequent optimization, the nucleic acid sequences encoding the initially selected (poly)peptides can optionally be used without sequencing. Optimization is performed by repeating the replacement of sub-sequences by different sequences, preferably by random sequences, and the screening step one or more times.

The desired property the (poly)peptides are screened for is preferably, but not exclusively, selected from the group of optimized affinity or specificity for a target molecule, optimized enzymatic activity, optimized expression yields, optimized stability and optimized solubility.

.oem.i In one embodiment, the cleavage sites flanking the sub-sequences are sites recognized and cleaved by restriction enzymes, with recognition and cleavage sequences being either identical or different, the restricted sites either having blunt or sticky ends.

The length of the sub-elements is preferably, but not exclusively ranging between 1 amino acid, such as one residue in the active site of an enzyme or a structuredetermining residue, and 150. amino acids, as for whole protein domains. Most preferably, the length ranges between 3 and 25 amino acids, such as most commonly found in CDR loops of antibodies.

The nucleic acid sequences could be RNA or, preferably, DNA.

-7- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 In one embodiment, the (poly)peptides have an arrino acid pattern characteristic of a particular species. This can for example be achieved by deducing the consensus sequences from a collection of homologous proteins of just one species, most preferably from a collection of human proteins. Since the (poly)peptides comprising consensus sequences are artificial, they have to be compared to the protein sequence(s) having the closest similarity to ensure the presence of said characteristic amino acid pattern.

In one embodiment, the invention provides for the creation of libraries of (poly)peptides comprising at least part of members or derivatives of the immunoglobulin superfamily, preferably of member or derivatives of the immnoglobulins. Most preferably, the invention provides for the creation of libraries of human antibodies, wherein said (poly)peptides are or are derived from heavy or light chain variable regions wherein said structural sub-elements are framework regions (FR) 1, 2, 3, or 4 or complementary determining regions (CDR) 1, 2, or 3. In a first step, a database of published antibody sequences of human origin is established where the antibody sequences are aligned to each other. The database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold of CDR loops (as determined by analysis of antibody structures). For each of the subgroups a consensus sequence is deduced which represents the members of this subgroup; the complete collection of consensus sequences represent therefore the complete structural repertoire of human antibodies.

These artificial genes are then constructed e.g. by total gene synthesis or by the use of synthetic genetic subunits. These.genetic subunits correspond to structural subelements on the (poly)peptide level. On the DNA level, these genetic subunits are defined by cleavage sites at the start and the end of each of the sub-elements, which are unique in the vector system. All genes which are members of the collection of consensus sequences are constructed such that they contain a similar pattern of corresponding genetic sub-sequences. Most preferably, said (poly)peptides are or are derived from the HuCAL consensus genes: VK1, VK2, VK3, VK4, VX1, VX2, V?,3, VH1A, VH1B, VH2, VH3, VH4, VH5, VH6, CK, Ck, CH1 or any combination of said HuCAL consensus genes.

This collection of DNA molecules can then be used to create libraries of antibodies or antibody fragments, preferably Fv, disulphide-linked Fv, single-chain Fv (scFv), or Fab fragments, which may be used as sources of specificities against new target antigens. Moreover, the affinity of the antibodies can be optimized using pre-built library cassettes and a general procedure. The invention provides a method for identifying one or more genes encoding one or more antibody fragments which -8- SUBSTITUTE SHEET (RULE 26)

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WO 97/08320 PCT/EP96/03647 binds to a target, comprising the steps of ,xpessir.g the antibody fragments, and then screening them to isolate one or more antibody fragments which bind to a given target molecule. Preferably, an scFv fragment library comprising the combination of HuCAL VH3 and HuCAL VX2 consensus genes and at least a random sub-sequence encoding the heavy chain CDR3 sub-element is screened for binding antibodies. If necessary, the modular design of the genes can then be used to excise from the genes encoding the antibody fragments one or more genetic subsequences encoding structural sub-elements, and replacing them by one or more second sub-sequences encoding structural sub-elements. The expression and screening steps can then be repeated until an antibody having the desired affinity is generated.

Particularly preferred is a method in which one or more of the genetic subunits (e.g.

the CDRs) are replaced by a random collection of sequences (the library) using the said cleavage sites. Since these cleavage sites are unique in the vector system and (ii) common to all consensus genes, the same (pre-built) library can be inserted into all artificial antibody genes. The resulting library is then screened against any chosen antigen. Binding antibodies are selected, collected and used as starting material for the next library. Here, one or more of the remaining genetic subunits are randomized as described above.

A further embodiment of the present invention relates to fusion proteins by providing for a DNA sequence which encodes both the (poly)peptide, as described above, as well as an additional moiety. Particularly preferred are moieties which have a useful therapeutic function. For example, the additional moiety may be a toxin molecule which is able to kill cells (Vitetta et al., 1993). There are numerous examples of such toxins, well known to the one skilled in the art, such as the bacterial toxins Pseudomonas exotoxin A, and diphtheria toxin, as well as the plant toxins ricin, abrin, modeccin, saporin, and gelonin. By fusing such a toxin for example to an antibody fragment, the toxin can be targeted to, for example, diseased cells, and thereby have a beneficial therapeutic effect. Alternatively, the additional moiety may be a cytokine, such as IL-2 (Rosenberg Lotze, 1986), which has a particular effect (in this case a T-cell proliferative effect) on a family of cells. In a further embodiment, the additional moiety may confer on its (poly)peptide partner a means of detection and/or purification. For example, the fusion protein could comprise the modified antibody fragment and an enzyme commonly used for detection purposes, such as alkaline phosphatase (Blake et al., 1984). There are numerous other moieties which can be used as detection or purification tags, which are well known to the practitioner skilled in the art. Particularly preferred are peptides comprising at least five histidine residues (Hochuli et al., 1988), which are able to bind to metal ions, -9- SUBSTITUTE SHEET (RULE 26) and can therefore be used for the purification of the protein to which they are fused (Lindner et al., 1992). Also provided for by the invention are additional moieties such as the commonly used C-myc and FLAG tags (Hopp et al., 1988; Knappik PlIckthun, 1994).

By engineering one or more fused additional domains, antibody fragments or any other (poly)peptide can be assembled into larger molecules which also fall under the scope of the present invention. For example, mini-antibodies (Pack, 1994) are dimers comprising two antibody fragments, each fused to a self-associating dimerization domain. Dimerization domains which are particularly preferred include those derived from a leucine zipper (Pack Plickthun, 1992) or helix-turn-helix motif (Pack et al., 1993).

All of the above embodiments of the present invention can be effected using standard techniques of molecular biology known to anyone skilled in the art.

In a further embodiment, the random collection of sub-sequences (the library) is inserted into a singular nucleic acid sequence encoding one (poly)peptide, thus creating a (poly)peptide library based on one universal framework. Preferably a random collection of CDR sub-sequences is inserted into a universal antibody framework, for example into the HuCAL H3k2 single-chain Fv fragment described above.

In further embodiments, the invention provides for nucleic acid sequence(s), vector(s) containing the nucleic acid sequence(s), host cell(s) containing the vector(s), and (poly)peptides, obtainable according to the methods described above.

In a further preferred embodiment, the invention provides for modular vector systems comprising a cleavage site suitable for efficient ligation without modification with the modular nucleic acid sequences encoding the (poly)peptides. The modules of the vectors are flanked by restriction sites unique within the vector system and essentially unique with respect to the restriction sites incorporated into the nucleic acid sequences encoding the (poly)peptides, except for example the restriction sites necessary for cloning the nucleic acid sequences into the vector. The list of vector modules comprises origins of single-stranded replication, origins of double-stranded 5 replication for high- and low copy number plasmids, promotor/operator, repressor or terminator elements, resistance genes, potential recombination sites, gene III for display on filamentous phages, signal sequences, purification and detection tags, S and sequences of additional moieties.

H:\Gabriela\Keep\Speci\13686-01.doc 26/05/2003 WO 97/08320 PCT/EP96/03647 The vectors are preferably, but not exclusively, oxp:ession voctors or vectors suitable for expression and screening of libraries.

In another embodiment, the invention provides for a kit, comprising one or more of the list of nucleic acid sequence(s), recombinant vector(s), (poly)peptide(s), and vector(s) according to the methods described above, and suitable host cell(s) for producing the (poly)peptide(s).

In a preferred embodiment, the invention provides for the creation of libraries of human antibodies. In a first step, a database of published antibody sequences of human origin is established: The database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold (as determined by analysis of antibody structures). For each of the subgroups a consensus sequence is deduced which represents the members of this subgroup; the complete collection of consensus sequences represent therefore the complete structural repertoire of human antibodies.

These artificial genes are then constructed by the use of synthetic genetic subunits.

These genetic. subunits correspond to structural sub-elements on the protein level.

On the DNA level, these genetic subunits are defined by cleavage sites at the start and the end of each of the subelements, which are unique in the vector system. All genes which are members of the collection of consensus sequences are constructed such that they contain a similar pattern of said genetic subunits.

This collection of DNA molecules can then be used to create libraries of antibodies which may be used as sources of specificities against new target antigens.

S* Moreover, the affinity of the antibodies can be optimised using pre-built library cassettes and a general procedure. The invention provides a method for identifying one or more genes encoding one or more antibody fragments which binds to a :.target, comprising the steps of expressing the antibody fragments, and then screening them to isolate one or more antibody fragments which bind to a given target molecule. If necessary, the modular design of the genes can then be used to excise from the genes encoding the antibody fragments one or more genetic subsequences encoding structural sub-elements, and replacing them by one or more second sub-sequences encoding structural sub-elements. The expression and screening steps can then be repeated until an antibody having the desired affinity is generated.

-11- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Particularly preferred is a method in which one or more of the genetic subuiiits (e.g.

the CDR's) are replaced by a random collection of sequences (the library) using the said cleavage sites. Since these cleavage sites are unique in the vector system and (ii) common to all consensus genes, the same (pre-built) library can be inserted into all artificial antibody genes. The resulting library is then screened against any chosen antigen. Binding antibodies are eluted, collected and used as starting material for the next library. Here, one or more of the remaining genetic subunits are randomised as described above.

9 9 9 9 9 9 9 999999 -12- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Definitions Protein: The term protein comprises monomeric polypeptide chains as well as homo- or heteromultimeric complexes of two or more polypeptide chains connected either by covalent interactions (such as disulphide bonds) or by non-covalent interactions (such as hydrophobic or electrostatic interactions).

Analysis of homologous proteins: The amino acid sequences of three or more proteins are aligned to each other (allowing for introduction of gaps) in a way which maximizes the correspondence between identical or similar amino acid residues at all positions. These aligned sequences are termed homologous if the percentage of the sum of identical and/or similar residues exceeds a defined threshold. This threshold is commonly regarded by those skilled in the art as being exceeded when at least 15% of the amino acids in the aligned genes are identical, and at least 30% are similar. Examples for families of homologous proteins are: immunoglobulin superfamily, scavenger receptor superfamily, fibronectin superfamilies type II and III), complement control protein superfamily, cytokine receptor superfamily, cystine knot proteins, tyrosine kinases, and numerous other examples well known to one of ordinary skill in the art.

Consensus sequence: 09...0 Using a matrix of at least three aligned amino acid sequences, and allowing for gaps in the alignment, it is possible to determine the most frequent amino acid i residue at each position. The consensus sequence is that sequence which comprises the amino acids which are most frequently represented at each position.

In the event that two or more amino acids are equally represented at a single position, the consensus sequence includes both or all of those amino acids.

Removing unfavorable interactions: The consensus sequence is per se in most cases artificial and has to be analyzed in order to change amino acid residues which, for example, would prevent the resulting molecule to adapt a functional tertiary structure or which would block the interaction with other (poly)peptide chains in multimeric complexes. This can be done either by building a three-dimensional model of the consensus sequence using known related structures as a template, and identifying amino acid residues within the model which may interact unfavorably with each other, or (ii) analyzing the matrix of aligned amino acid sequences in order to detect combinations of amino -13- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP9/03647 acid residues within the sequences which frequent!y occur together in one sequence and are therefore likely to interact with each other. These probable interaction-pairs are then tabulated and the consensus is compared with these "interaction maps". Missing or wrong interactions in the consensus are repaired accordingly by introducing appropriate changes in amino acids which minimize unfavorable interactions.

Identification of structural sub-elements: Structural sub-elements are stretches of amino acid residues within a protein/(poly)peptide which correspond to a defined structural or functional part of the molecule. These can be loops CDR loops of an antibody) or any other secondary or functional structure within the protein/(poly)peptide (domains, ahelices, B-sheets, framework regions of antibodies, etc.). A structural sub-element can be identified using known structures of similar or homologous (poly)peptides, or by using the above mentioned matrices of aligned amino acid sequences. Here the variability at each position is the basis for determining stretches of amino acid residues which belong to a structural sub-element hypervariable regions of an antibody).

Sub-seuece: A sub-sequence is defined as a genetic module which is flanked by unique cleavage sites and encodes at least one structural sub-element. It is not necessarily identical to a structural sub-element.

Cleavage site: A short DNA sequence which is used as a specific target for a reagent which cleaves DNA in a sequence-specific manner restriction endonucleases).

Comoatible cleavage sites: Cleavage sites are compatible with each other, if they can be efficiently ligated without modification and, preferably, also without adding an adapter molecule..

Unique cleavage sites: A cleavage site is defined as unique if it occurs only once in a vector containing at least one of the genes of interest, or if a vector containing at least one of the genes of interest could be treated in a way that only one of the cleavage sites could be used by the cleaving agent.

-14- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Corresponding (polvypeptide sequences: Sequences deduced from the same part of one group of homologous proteins are called corresponding (poly)peptide sequences.

Common cleavage sites: A cleavage site in at least two corresponding sequences, which occurs at the same functional position which flanks a defined sub-sequence), which can be hydrolyzed by the same cleavage tool and which yields identical compatible ends is termed a common cleavage site.

Excising genetic sub-sequences: A method which uses the unique cleavage sites and the corresponding cleavage reagents to cleave the target DNA at the specified positions in order to isolate, remove or replace the genetic sub-sequence flanked by these unique cleavage sites.

Exchanging genetic sub-sequences: A method by which an existing sub-sequence is removed using the flanking cleavage sites of this sub-sequence, and a new sub-sequence or a collection of sub-sequences, which contain ends compatible with the cleavage sites thus created, is inserted.

Expression of genes: The term expression refers to in vivo or in vitro processes, by which the information of a gene is transcribed into mRNA and then translated into a protein/(poly)peptide.

Thus, the term expression refers to a process which occurs inside cells, by which the information of a gene is transcribed into mRNA and then into a protein. The term expression also includes all events of post-translational modification and transport, which are necessary for the (poly)peptide to be functional.

Screening of protein/(Dolvy)eptide libraries: Any method which allows isolation of one or more proteins/(poly)peptides having a desired property from other proteins/(poly)peptides within a library.

Amino acid pattern characteristic for a species: A (poly)peptide sequence is assumed to exhibit an amino acid pattern characteristic for a species if it is deduced from a collection of homologous proteins from just this species.

SUBSTITUTE SHEET (RULE 26) A method by which an existing sub-sequence is removed using the flanking cleavage sites of this sub-sequence, and a new sub-sequenceor collection of sub-sequences, which contains ends compatible with the cleavage sites thus created, is inserted.

Assembling of genetic sequences: Any process which is used to combine synthetic or natural genetic sequences in a specific manner in order to get longer genetic sequences which contain at least parts of the used synthetic or natural genetic sequences.

Analysis of homoloqous genes: The corresponding amino acid sequences of two or more genes are aligned to each other in a way which maximizes the correspondence between identical or similar amino acid residues at all positions. These aligned sequences are termed homologous if the percentage of the sum of identical and/or similar residues exceeds a defined threshold. This threshold is commonly regarded by those skilled in the art as being exceeded when at least 15 per cent of the amino acids in the aligned genes are identical, and at least 30 per cent are similar.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

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ooooo -16- 17 All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

e H:\Gabriela\Keep\SpecR\13686-01 .doc 26/05/2003 WO 97/08320 PCT/EP96/0 3 64 7 Legends to Figures and Tables Fig. 1: Flow chart outlining the process of construction of a synthetic human antibody library based on consensus sequences.

Fig. 2: Alignment of consensus sequences designed for each subgroup (amino acid residues are shown with their standard one-letter abbreviation).

(A)

kappa sequences, lambda sequences and heavy chain sequences. The positions are numbered according to Kabat (1991). In order to maximize homology in the alignment, gaps have been introduced in the sequence at certain positions.

Fig. 3: Gene sequences of the synthetic V kappa consensus genes. The corresponding amino acid sequences (see Fig. 2) as well as the unique cleavage sites are also shown.

Fig. 4: Gene sequences of the synthetic V lambda consensus genes. The corresponding amino acid sequences (see Fig. 2) as well as the unique cleavage sites are also shown.

Fig. 5: Gene sequences of the synthetic V heavy chain consensus genes. The corresponding amino acid sequences (see Fig. 2) as well as the unique cleavage sites are also shown.

Fig. 6: Oligonucleotides used for construction of the consensus genes. The oligos are named according to the corresponding consensus gene, e.g.

the gene VK1 was constructed using the six oligonucleotides 01K1 to 01K6. The oligonucleotides used for synthesizing the genes encoding theconstant domains CK (OCLK1 to 8) and CH1 (OCHI to 8) are also shown.

F.

Fig. 7A/B: Sequences of the synthetic genes encoding the constant domains

C,

and CH1 The corresponding amino acid sequences as well as :i F unique cleavage sites introduced in these genes are also shown. Fig. 7C: Functional map and sequence of module M24 comprising the synthetic C. gene segment (huCL lambda).

Fig. 7D: Oligonucleotides used for synthesis of module M24.

Fig. 8: Sequence and restriction map of the synthetic gene encoding the consensus single-chain fragment VH3-VK2. The signal sequence (amino acids 1 to 21) was derived from the E. coli phoA gene (Skerra -18- SUBSTTUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Plickthun, 1988). Between the phoA signal scqu2nce rnd the VH3 domain, a short sequence stretch encoding 4 amino acid residues (amino acid 22 to 25) has been inserted in order to allow detection of the singlechain fragment in Western blot or ELISA using the monoclonal antibody M1 (Knappik Pluckthun, 1994). The last 6 basepairs of the sequence were introduced for cloning purposes (EcoRI site).

Fig. 9: Plasmid map of the vector plG10.3 used for phage display of the H3K2 scFv fragment. The vector is derived from pIG10 and contains the gene for the lac operon repressor, lad, the artificial operon encoding the H3K2gene3ss fusion under control of the lac promoter, the Ipp terminator of transcription, the single-strand replication origin of the E. coli phage f1 (F1_ORI), a gene encoding P-lactamase (bla) and the ColEI derived origin of replication.

Fig. 10: Sequencing results of independent clones from the initial library, translated into the corresponding amino acid sequences. Amino acid sequence of the VH3 consensus heavy chain CDR3 (position 93 to 102, Kabat numbering). Amino acid sequences of 12 clones of the library. Amino acid sequences of 11 clones of the 15-mer library, single base deletion.

SFig. 11: Expression test of individual library members. Expression of 9 independent clones of the 10-mer library. Expression of 9 Sindependent clones of the 15-mer library. The lane designated with M contains the size marker. Both the gp3-scFv fusion and the scFv monomer are indicated.

Fig. 12: Enrichment of specific phage antibodies during the panning against FITC- BSA. The initial as well as the subsequent fluorescein-specific sublibraries were panned against the blocking buffer and the ratio of the e phage eluted from the FITC-BSA coated well vs. that from the powder milk coated well from each panning round is presented as the ,,specificity factor".

Fig. 13: Phage ELISA of 24 independent clones after the third round of panning i tested for binding on FITC-BSA.

Fig. 14: Competition ELISA of selected FITC-BSA binding clones. The ELISA signals of scFv binding without inhibition are taken as 100%.

Fig. 15: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against FITC-BSA, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering).

-19- SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 Fig. 16: Coomassie-Blue stained SDS-PAGE of the purif:od anti-flucrecPin scFv fragments: M: molecular weight marker, A: total soluble cell extract after induction, B: fraction of the flow-through, C, D and E: purified scFv fragments 1HA-3E4, 1HA-3E5 and 1HA-3E10, respectively.

Fig. 17: Enrichment of specific phage antibodies during the panning against 3estradiol-BSA, testosterone-BSA, BSA, ESL-1, interleukin-2, lymphotoxin-B, and LeY-BSA after three rounds of panning.

Fig. 18: ELISA of selected ESL-1 and B-estradiol binding clones Fig. 19: Selectivity and cross-reactivity of HuCAL antibodies: in the diagonal specific binding of HuCAL antibodies can be seen, off-diagonal signals show non-specific cross-reactivity.

Fig. 20: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against 1-estradiol-BSA, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering). One clone is derived from the 10mer library.

Fig. 21: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against testosterone-BSA, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering).

ig. 22: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against lymphotoxin-B, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering). One clone comprises a 14mer CDR, presumably introduced by incomplete coupling of the trinucleotide mixture during oligonucleotide synthesis.

.0 Fig. 23: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against ESL-1, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering). Two clones are derived from the 10mer library. One clone comprises a 16mer CDR, presumably introduced by chain elongation during oligonucleotide synthesis using trinucleotides.

Fig. 24: Sequencing results of the heavy chain CDR3s of independent clones after 3 rounds of panning against BSA, translated into the corresponding amino acid sequences (position 93 to 102, Kabat numbering).

Fig. 25: Schematic representation of the modular pCAL vector system.

Fig. 25a: List of restriction sites already used in or suitable for the modular HuCAL genes and pCAL vector system.

Fig. 26: List of the modular vector elements for the pCAL vector series: shown are only those restriction sites which are part of the modular system.

SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Fig. 27: Functional map and sequence of the multi-cloning site module ,MCS) Fig. 28: Functional map and sequence of the pMCS cloning vector series.

Fig. 29: Functional map and sequence of the pCAL module M1 (see Fig. 26).

Fig. 30: Functional map and sequence of the pCAL module M7-111 (see Fig. 26).

Fig. 31: Functional map and sequence of the pCAL module M9-ll (see Fig. 26).

Fig. 32: Functional map and sequence of the pCAL module Ml1-11 (see Fig. 26).

Fig. 33: Functional map and sequence of the pCAL module M14-Ext2 (see Fig.

26).

Fig. 34: Functional map and sequence of the pCAL module M17 (see Fig. 26).

Fig. 35: Functional map and sequence of the modular vector pCAL4.

Fig. 35a:Functional maps and sequences of additional pCAL modules (M2, M3, M71, M711, M8, M1011, M11II, M12, M13, M19, M20, M21, M41) and of lowcopy number plasmid vectors (pCALO1 to pCALO3).

Fig. 35b:List of oligonucleotides and primers used for synthesis of pCAL vector modules.

Fig. 36: Functional map and sequence of the B-lactamase cassette for replacement of CDRs for CDR library cloning.

Fig. 37: Oligo and primer design for VK CDR3 libraries Fig. 38: Oligo and primer design for VX CDR3 libraries Fig. 39: Functional map of the pBS13 expression vector series.

Fig. 40: Expression of all 49 HuCAL scFvs obtained by combining each of the 7 VH genes with each of the 7 VL genes (pBS13, 30°C): Values are given for the percentage of soluble vs. insoluble material, the total and the soluble amount compared to the combination H3K2, which was set to 100%. In addition, the corresponding values for the McPC603 scFv are given.

lo Table 1: Summary of human immunoglobulin germline sequences used for computing the germline membership of rearranged sequences. kappa sequences, lambda sequences and heavy chain sequences. (1) The germline name used in the various calculations, the references number for the corresponding sequence (see appendix for sequence related citations), the family where each sequence belongs to and the various names found in literature for germline genes with identical amino acid sequences.

Table 2: Rearranged human sequences used for the calculation of consensus sequences. kappa sequences, lambda sequences and heavy chain sequences. The table summarized the name of the sequence -21- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/036 4 7 the length of the sequence in amino acids th2 gsrmlin? fami!y as well as the computed germline counterpart The number of amino acid exchanges between the rearranged sequence and the germline sequence is tabulated in and the percentage of different amino acids is given in Column gives the references number for the corresponding sequence (see appendix for sequence related citations).

Table 3: Assignment of rearranged V sequences to their germline counterparts.

kappa sequences, lambda sequences and heavy chain sequences. The germline genes are tabulated according to their family and the number of rearranged genes found for every germline gene is given in Table 4: Computation of the consensus sequence of the rearranged V kappa sequences. V kappa subgroup 1, V kappa subgroup 2, V kappa subgroup 3 and V kappa subgroup 4. The number of each amino acid found at each position is tabulated together with the statistical analysis of the data. Amino acids are given with their standard oneletter abbreviations (and B means D or N, Z means E or Q and X means any amino acid). The statistical analysis summarizes the number of sequences found at each position the number of occurrences of the most common amino acid the amino acid residue which is most common at this position the relative frequency of the occurrence of the most common amino acid and the number of different amino acids found at each position Table 5: Computation of the consensus sequence of the rearranged V lambda sequences. V lambda subgroup 1, V lambda subgroup 2, and V lambda subgroup 3. The number of each amino acid found at each position is tabulated together with the statistical analysis of the data.

Abbreviations are the same as in Table 4.

Table 6: Computation of the consensus sequence of the rearranged V heavy chain sequences. V heavy chain subgroup 1A, V heavy chain subgroup 1B, V heavy chain subgroup 2, V heavy chain subgroup 3, V heavy chain subgroup 4, V heavy chain subgroup and V heavy chain subgroup 6. The number of each amino acid found at each position is tabulated together with the statistical analysis of the data. Abbreviations are the same as in Table 4.

-22- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Examples Example 1: Design of a Synthetic Human Combinatorial Antibody Library (HuCAL) The following example describes the design of a fully synthetic human combinatorial antibody library (HuCAL), based on consensus sequences of the human immunoglobulin repertoire, and the synthesis of the consensus genes. The general procedure is outlined in Fig. 1.

1.1 Sequence database 1.1.1 Collection and alignment of human immunoglobulin sequences In a first step, sequences of variable domains of human immunoglobulins have been collected and divided into three sub bases: V heavy chain V kappa (VK) and V lambda For each sequence, the gene sequence was then translated into the corresponding amino acid sequence. Subsequently, all amino acid sequences were aligned according to Kabat et al. (1991). In the case of VX sequences, the numbering system of Chuchana et al. (1990) was used. Each of the three main databases was then divided into two further sub bases: the first sub base contained all sequences derived from rearranged V genes, where more than 70 positions of the sequence were known. The second sub base contained all germline gene segments (without the D- and J- minigenes; pseudogenes with internal stop codons were also removed). In all cases, where germline sequences with identical amino acid sequence but different names were found, only one sequence was used (see Table The final databases of rearranged sequences contained 386, 149 and 674 entries for VK, VX and VH, respectively. The final databases of germline sequences contained 48, 26 and 141 entries for VK, VX and VH, respectively.

1.1.2 Assignment of sequences to subgroups The sequences in the three germline databases where then grouped according to sequence homology (see also Tomlinson et al., 1992, Williams Winter, 1993, and Cox et al., 1994). In the case of VK, 7 families could be established. V. was divided into 8 families and VH into 6 families. The VH germline genes of the VH7 family (Van Dijk et al., 1993) were grouped into the VH1 family, since the genes of the two families are highly homologous. Each family contained different numbers of germline genes, varying from 1 (for example VH6) to 47 (VH3).

-23- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 1.2 Analysis of sequences 1.2.1 Computation of germline membership For each of the 1209 amino acid sequences in the databases of rearranged genes, the nearest germline counterpart, i.e. the germline sequence with the smallest number of amino acid differences was then calculated. After the germline counterpart was found, the number of somatic mutations which occurred in the rearranged gene and which led to amino acid exchanges could be tabulated. In 140 cases, the germline counterpart could not be calculated exactly, because more than one germline gene was found with an identical number of amino acid exchanges.

These rearranged sequences were removed from the database. In a few cases, the number of amino acid exchanges was found to be unusually large (>20 for VL and for VH), indicating either heavily mutated rearranged genes or derivation from germline genes not present in the database. Since it was not possible to distinguish between these two possibilities, these sequences were also removed from the database. Finally, 12 rearranged sequences were removed from the database because they were found to have very unusual CDR lengths and composition or unusual amino acids at canonical positions (see below). In summary, 1023 rearranged sequences out of 1209 could be clearly assigned to their germline counterparts (see Table 2).

After this calculation, every rearranged gene could be arranged in one of the families established for the germline genes. Now the usage of each germline gene, i.e. the number of rearranged genes which originate from each germline gene, could be calculated (see Table It was found that the usage was strongly biased towards a subset of germline genes, whereas most of the germline genes were not present *i as rearranged genes in the database and therefore apparently not used in the immune system (Table This observation had already been reported in the case of VK (Cox, et al., 1994). All germline gene families, where no or only very few rearranged counterparts could be assigned, were removed from the database, leaving 4 VK, 3 and 6 VH families.

S

1.2.2 Analysis of CDR conformations The conformation of the antigen binding loops of antibody molecules, the CDRs, is strongly dependent on both the length of the CDRs and the amino acid residues located at the so-called canonical positions (Chothia Lesk, 1987). It has been found that only a few canonical structures exist, which determine the structural -24.

SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 repertoire of the immunoglobulin variable domains (Chothia et 1989). The canonical amino acid positions can be found in CDR as well as framework regions.

The 13 used germline families defined above (7 VL and 6 VH) were now analyzed for their canonical structures in order to define the structural repertoire encoded in these families.

In 3 of the 4 VK families (VK1; 2 and one different type of CDR1 conformation could be defined for every family. The family VK3 showed two types of CDR1 conformation: one type which was identical to V1 and one type only found in Vi3.

All VK CDR2s used the same type of canonical structure. The CDR3 conformation is not encoded in the germline gene segments. Therefore, the 4 VK families defined by sequence homology and usage corresponded also to 4 types of canonical structures found in VK germline genes.

The 3 V% families defined above showed 3 types of CDR1 conformation, each family with one unique type. The VX1 family contained 2 different CDR1 lengths (13 and 14 amino acids), but identical canonical residues, and it is thought that both lengths adopt the same canonical conformation (Chothia Lesk, 1987). In the CDR2 of the used VX germlines, only one canonical conformation exists, and the CDR3 conformation is not encoded in the germline gene segments. Therefore, the 3 VX families defined by sequence homology and usage corresponded also to 3 types of canonical structures.

The structural repertoire of the human VH sequences was analyzed in detail by Chothia et al., 1992. In total, 3 conformations of CDR1 (H1-1, H1-2 and H1-3) and 6 conformations of CDR2 (H2-1. H2-2, H2-3, H2-4, H2-5 and H2-x) could be defined.

Since the CDR3 is encoded in the D- and J-minigene segments, no particular canonical residues are defined for this CDR.

All the members of the VH1 family defined above contained the CDR1 conformation H1-1, but differed in their CDR2 conformation: the H2-2 conformation was found in 6 germline genes, whereas the conformation H2-3 was found in 8 germline genes.

Since the two types of CDR2 conformations are defined by different types of amino acid at the framework position 72. the VH1 family was divided into two subfamilies: VH1A with CDR2 conformation H2-2 and VH1B with the conformation H2-3. The members of the VH2 family all had the conformations H1-3 and H2-1 in CDR1 and CDR2, respectively. The CDR1 conformation of the VH3 members was found in all cases to be H1-1, but 4 different types were found in CDR2 (H2-1, H2-3, H2-4 and H2-x). In these CDR2 conformations, the canonical framework residue 71 is always SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 defined by an arginine. Therefore, it was not necessary to civide the V\X3 family into subfamilies, since the 4 types of CDR2 conformations were defined solely by the CDR2 itself. The same was true for the VH4 family. Here, all 3 types of CDR1 conformations were found, but since the CDR1 conformation was defined by the CDR itself (the canonical framework residue 26 was found to be glycine in all cases), no subdivisions were necessary. The CDR2 conformation of the VH4 members was found to be H2-1 in all cases. All members of the VH5 family were found to have the conformation H1-1 and H2-2, respectively. The single germline gene of the VH6 family had the conformations H1-3 and H2-5 in CDR1 and CDR2, respectively.

In summary, all possible CDR conformations of the VK and VK genes were present in the 7 families defined by sequence comparison. From the 12 different CDR conformations found in the used VH germline genes, 7 could be covered by dividing the family VH1 into two subfamilies, thereby creating 7 VH families. The remaining CDR conformations (3 in the VH3 and 2 in the VH4 family) were defined by the CDRs themselves and could be created during the construction of CDR libraries.

Therefore, the structural repertoire of the used human V genes could be covered by 49 (7 x 7) different frameworks.

1.2.3 Computation of consensus sequences The 14 databases of rearranged sequences (4 VK, 3 VX and 7 VH) were used to compute the HuCAL consensus sequences of each subgroup (4 HuCAL- VK, 3 HuCAL- Vk, 7 HuCAL- VH, see Table 4, 5 and This was done by counting the number of amino acid residues used at each position (position variability) and subsequently identifying the amino acid residue most frequently used at each position. By using the rearranged sequences instead of the used germline sequences for the calculation of the consensus, the consensus was weighted according to the frequency of usage. Additionally, frequently mutated and highly conserved positions could, be identified. The consensus sequences were crosschecked with the consensus of the germline families to see whether the rearranged sequences were biased at certain positions towards amino acid residues which do not occur in the collected germline sequences, but this was found not to be the case.

Subsequently, the number of differences of each of the 14 consensus sequences to each of the germline sequences found in each specific family was calculated. The overall deviation from the most homologous germline sequence was found to be 2.4 amino acid residues ensuring that the "artificial" consensus sequences -26- SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 can still be considered as truly human sequences as far as immunol3cnicity is concerned.

1.3 Structural analysis So far, only sequence information was used to design the consensus sequences.

Since it was possible that during the calculation certain artificial combinations of amino acid residues have been created, which are located far away in the sequence but have contacts to each other in the three dimensional structure, leading to destabilized or even misfolded frameworks, the 14 consensus sequences were analyzed according to their structural properties.

It was rationalized that all rearranged sequences present in the database correspond to functional and therefore correctly folded antibody molecules. Hence, the most homologous rearranged sequence was calculated for each consensus sequence. The positions where the consensus differed from the rearranged sequence were identified as potential "artificial residues" and inspected.

The inspection itself was done in two directions. First, the local sequence stretch around each potentially "artificial residue" was compared with the corresponding stretch of all the rearranged sequences. If this stretch was found to be truly artificial, i.e. never occurred in any of the rearranged sequences, the critical residue was converted into the second most common amino acid found at this position and analyzed again. Second, the potentially "artificial residues" were analyzed for their long range interactions. This was done by collecting all available structures of human antibody variable domains from the corresponding PDB files and calculating for every structure the number and type of interactions each amino acid residue established to each side-chain. These "interaction maps" were used to analyze the probable side-chain/side-chain interactions of the potentially "artificial residues". As a result of this analysis, the following residues were exchanged (given is the name of the gene, the position according to Kabat's numbering scheme, the amino acid found at this position as the most abundant one and the amino acid which was used ***instead): VH2: ST SVK1: N34A, VK3: GgA, D 60 A, R 77

S

V,3: V 7 8

T

-27- SUBSTIUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96103647 1.4 Design of CDR sequences The process described above provided the complete consensus sequences derived solely from the databases of rearranged sequences. It was rationalized that the ODRi and CDR2 regions should be takeh from the databases of used germline sequences, since the CDRs of rearranged and mutated sequences are biased towards their particular antigens. Moreover, the germline CDR sequences are known to allow binding to a variety of antigens in the primary immune response, where only CDR3 is varied. Therefore, the consensus .CDRs obtained from the calculations described above were replaced by germline CDRs in the case of VH and VK. In the case of VX, a few amino acid exchanges were introduced in some of the chosen germline CORs in order to avoid possible protease cleavage sites as well as possible structural constraints.

The CORs of following germline genes have been chosen: HuCAL gene HuCAL-VH1 A HuCAL-VH1 B HuCAL-VH2 HuCAL-VH3 HuCAL-VH4 HuCAL-VH5 HuCAL-VH6 HuCAL-VK 1 HuCAL-VK2 HuCAL-VK3 HuCAL-VK4 HuCAL-VX1 H uCA L-VX2 HuCAL-V?,3 CDR1 VH1-12-1 VH-1-13-16 VH2-31-lO,-1 1,-i 2-13 VH3-1 0.

VH-4-11-7 to 14 VH5-1 2-1 ,-2 VH6-3 5-1 VK1-14,-15 VK2-6 VK3-1 ,-4 VW4-1 HUMLV1 17,DPL5 DPL1 1 ,DPL1 2 DP L23 M2 VH 11-12-1 V~I- VH2-31 -3,-4 VH3- 13-8,-9,-1 0 VH4-1 1,-i 2,-i 4,-i 6 VH4-31 -17,-i 8,-i 9,-20 VH5-12-i ,-2 VH-6-35- 1 VK 1 VK2-6 VK3-4 VK4-1 DP-1 2 HUMLV318 -28- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 In the case of the CDR3s, any sequence could be chosen since these CDRs were planned to be the first to be replaced by oligonucleotide libraries. In order to study the expression and folding behavior of the consensus sequences in E. coli, it would be useful to have all sequences with the same CDR3, since the influence of the CDR3s on the folding behavior would then be identical in all cases. The dummy sequences QQHYTTPP and ARWGGDGFYAMDY were selected for the VL chains (kappa and lambda) and for the VH chains, respectively. These sequences are known to be compatible with antibody folding in E coli (Carter et al., 1992).

Gene design The final outcome of the process described above was a collection of 14 HuCAL amino acid sequences, which represent the frequently used structural antibody repertoire of the human immune system (see Figure These sequences were back-translated into DNA sequences. In a first step, the back-translation was done using only codons which are known to be frequently used in E. coli. These gene sequences were then used for creating a database of all possible restriction endonuclease sites, which could be introduced without changing the corresponding amino acid sequences. Using this database, cleavage sites were selected which were located at the flanking regions of all sub-elements of the genes (CDRs and framework regions) and which could be introduced in all HuCAL VH, VK or VX genes simultaneously at the same position. In a few cases it was not possible to find cleavage sites for all genes of a subgroup. When this happened, the amino acid sequence was changed, if this was possible according to the available sequence and structural information. This exchange was then analyzed again as described above. In total, the following 6 amino acid residues were exchanged during this design (given is the name of the gene, the position according to Kabat's numbering scheme, the amino acid found at this position as the most abundant one and the amino acid which was used instead): i VH2:

TQ

VH6: S,,G VK3: E,D, 1 58

V

VK4: K 2

,R

VX3:

T,,S

-29- SUBSTTUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 In one case (5'-end of VH framework 3) it was not possible to idontify a single cleavage site for all 7 VH genes. Two different type of cleavage sites were used instead: BstEll for HuCAL VH1A, VH1B, VH4 and VH5, and NspV for HuCAL VH2, VH3, VH4 and VH6.

Several restriction endonuclease sites were identified, which were not located at the flanking regions of the sub-elements but which could be introduced in every gene of a given group without changing the amino acid sequence. These cleavage sites were also introduced in order to make the system more flexible for further improvements. Finally, all but one remaining restriction endonuclease sites were removed in every gene sequence. The single cleavage site, which was not removed was different in all genes of a subgroup and could be therefore used as a "fingerprint" site to ease the identification of the different genes by restriction digest.

The designed genes, together with the corresponding amino acid sequences and the group-specific restriction endonuclease sites are shown in Figure 3, 4 and respectively.

1.6 Gene synthesis and cloning The consensus genes were synthesized using the method described by Prodromou Pearl, 1992, using the oligonucleotides shown in Fig. 6. Gene segments encoding the human constant domains CK, CK and CH1 were also synthesized, based on sequence information given by Kabat et al., 1991 (see Fig. 6 and Fig. Since for both the CDR3 and the framework 4 gene segments identical sequences were chosen in all HuCAL V

K

VX and VH genes, respectively, this part was constructed only once, together with the corresponding gene segments encoding the constant domains. The PCR products were cloned into pCR-Script (Stratagene, Inc.) or pZErO-1 (Invitrogen, Inc.) and verified by sequencing.

Example 2: Cloning and Testing of a HuCAL-Based Antibody Library A combination of two of the synthetic consensus genes was chosen after construction to test whether binding antibody fragments can be isolated from a library based on these two consensus frameworks. The two genes were cloned as a single-chain Fv (scFv) fragment, and a VH-CDR3 library was inserted. In order to test the library for the presence of functional antibody molecules, a selection procedure SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 was carried out using the small hapten fluorescein bound to 8SA ,F17C-BSA) as antigen.

2.1 Cloning of the HuCAL VH3-Vk2 scFv fragment In order to test the design of the consensus genes, one randomly chosen combination of synthetic light and heavy gene (HuCAL-VK2 and HuCAL-VH3) was used for the construction of a single-chain antibody (scFv) fragment. Briefly, the gene segments encoding the VH3 consensus gene and the CH1 gene segment including the CDR3 framework 4 region, as well as the VK2 consensus gene and the CK gene segment including the CDR3 framework 4 region were assembled yielding the gene for the VH3-CH1 Fd fragment and the gene encoding the VK2-CK light chain, respectively. The CH1 gene segment was then replaced by an oligonucleotide cassette encoding a 20-mer peptide linker with the sequence AGGGSGGGGSGGGGSGGGGS. The two oligonucleotides encoding this linker were TCAGCGGGTGGCGGTTCTGGCGGCGGTGGGAGCGGTGGCGGTGGTTC- TGGCGGTGGTGGTTCCGATATCGGTCCACGTACGG-3' and

TGGACCGATATCGGAACCACCACCGCCAGAACCACCGCCACCGCTCCCACCGC

CGCCAGAACCGCCACCCGC-3', respectively. Finally, the HuCAL-VK2 gene was inserted via EcoRV and BsiWI into the plasmid encoding the HuCAL-VH3-linker fusion, leading to the final gene HuCAL-VH3-VK2, which encoded the two S• consensus sequences in the single-chain format VH-linker-VL. The complete coding sequence is shown in Fig. 8.

o 2.2 Construction of a monovalent phage-display phagemid vector oooo* plG10.3 Phagemid plG10.3 (Fig. 9) was constructed in order to create a phage-display system (Winter et al., 1994) for the H3K2 scFv gene. Briefly, the EcoRI/Hindll restriction fragment in the phagemid vector pIG10 (Ge et al., 1995) was replaced by the c-myc followed by an amber codon (which encodes an glutamate in the ambersuppresser strain XL1 Blue and a stop codon in the non-suppresser strain JM83) and a truncated version of the gene III (fusion junction at codon 249, see Lowman et al., 1991) through PCR mutagenesis.

-31- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 2.3 Construction of H-CDR3 libraries Heavy chain CDR3 libraries of two lengths (10 and 15 amino acids) were constructed using trinucleotide codon containing oligonucleotides (Virnekas et al., 1994) as templates and the oligonucleotides complementing the flanking regions as primers. To concentrate only on the CDR3 structures that appear most often in functional antibodies, we kept the salt-bridge of and in the CDR3 loop. For the 15-mer library, both phenylalanine and methionine were introduced at position 100 since these two residues were found to occur quite often in human CDR3s of this length (not shown). For the same reason, valine and tyrosine were introduced at position 102. All other randomized positions contained codons for all amino acids except cystein, which was not used in the trinucleotide mixture.

The CDR3 libraries of lengths 10 and 15 were generated from the PCR fragments using oligonucleotide templates 03HCDR103T

GATACGGCCGTGTATTA-

TTGCGCGCGT (TRI),GATTATTGGGGCCAAGGCACCCTG-3') and 03HCDR153T GTATTATTGCGCGCGT(TRI),o(TTT/ATG)GAT(GTT/TAT)TGGG- GCCAAGGCACCCTG-3'), and primers 03HCDR35 TTGC-3') and 03HCDR33 (5'-CAGGGTGCCTTGGCCCC-3'), where TRI are trinucleotide mixtures representing all amino acids without cystein, (TTT/ATG) and (GTT/TAT) are trinucleotide mixtures encoding the amino acids phenylalanine/methionine and valine/tyrosine, respectively. The potential diversity of these libraries was 4.7 x 10 7 and 3.4 x 10 0 for 10-mer and 15-mer library, respectively. The library cassettes were first synthesized from PCR amplification of the oligo templates in the presence of both primers: 25 pmol of the oligo template 03HCDR103T or 03HCDR153T, 50 pmol each of the primers 03HCDR35 and 03HCDR33, 20 nmol of dNTP, 10x buffer and 2.5 units of Pfu DNA polymerase (Stratagene) in a total volume of 100 itl for 30 cycles (1 minute at 92C, 1 minute at 62 0C and 1 minute at 720C). A hot-start procedure was used. The resulting mixtures were phenol-extracted, ethanol-precipitated and digested overnight with Eagl and Styl. The vector plG10.3-scH3K2cat, where the Eagl-Styl fragment in the vector plG10.3-scH3K2 encoding the H-CDR3 was replaced by the chloramphenicol acetyltransferase gene (cat) flanked with these two sites, was similarly digested. The digested vector (35 pg) was gel-purified and ligated with 100 pg of the library cassette overnight at 160C. The ligation mixtures were isopropanol precipitated, airdried and the pellets were redissolved in 100 lt of ddH20. The ligation was mixed with 1 ml of freshly prepared electrocompetent XL1 Blue on ice. 20 rounds of electroporation were performed and the transformants were diluted in SOC medium, shaken at 37°C for 30 minutes and plated out on large LB plates (Amp/Tet/Glucose) -32- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 at 37°C for 6-9 hrs. The number of transformants (library size) was 3.2x10 7 and 2.3x10 7 for the 10-mer and the 15-mer library, respectively. The colonies were suspended in 2xYT medium (Amp/Tet/Glucose) and stored as glycerol culture.

In order to test the quality of the initial library, phagemids from 24 independent colonies (12 from the 10-mer and 12 from the 15-mer library, respectively) were isolated and analyzed by restriction digestion and sequencing. The restriction analysis of the 24 phagemids indicated the presence of intact vector in all cases.

Sequence analysis of these clones (see Fig. 10) indicated that 22 out of 24 contained a functional sequence in their heavy chain CDR3 regions. 1 out of 12 clones of the 10-mer library had a CDR3 of length 9 instead of 10, and 2 out of 12 clones of the 15-mer library had no open reading frame, thereby leading to a nonfunctional scFv; one of these two clones contained two consecutive inserts, but out of frame (data not shown). All codons introduced were presented in an even distribution.

Expression levels of individual library members were also measured. Briefly, 9 clones from each library were grown in 2xYT medium containing glucose at 370C overnight. Next day, the cultures were diluted into fresh medium with Amp/Tet. At an OD 6 ,nm of 0.4, the cultures were induced with 1 mM of IPTG and shaken at RT overnight. Then the cell pellets were suspended in 1 ml of PBS buffer 1 mM of EDTA. The suspensions were sonicated and the supernatants were separated on an SDS-PAGE under reducing conditions, blotted on nylon membrane and detected with anti-FLAG M1 antibody (see Fig. 11). From the nine clones of the 10-mer library, all express the scFv fragments. Moreover, the gene III /scFv fusion proteins were present in all cases. Among the nine clones from the 15-mer library analyzed, 6/9 led to the expression of both scFv and the gene III/scFv fusion proteins. More importantly, all clones expressing the scFvs and gene Ill/scFv fusions gave rise to about the same level of expression.

2.4 Biopanning Phages displaying the antibody libraries were prepared using standard protocols.

Phages derived from the 10-mer library were mixed with phages from the library in a ratio of 20:1 (1x10'° cfu/well of the 10-mer and 5x10 8 cfu/well of the mer phages, respectively). Subsequently, the phage solution was used for panning in ELISA plates (Maxisorp, Nunc) coated with FITC-BSA (Sigma) at concentration of 100 pg/ml in PBS at 4"C overnight. The antigen-coated wells were blocked with 3% powder milk in PBS and the phage solutions in 1% powder milk were added to each -33- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 well and the plate was shaken at RT for 1 hr. The wells w&re then washed with PBST and PBS (4 times each with shaking at RT for 5 minutes). The bound phages were eluted with 0.1 M triethylamine (TEA) at RT for 10 minutes. The eluted phage solutions were immediately neutralized with 1/2 the volume of 1 M Tris-CI, pH 7.6.

Eluted phage solutions (ca. 450 pl) were used to infect 5 ml of XL1 Blue cells at 37"C for 30 min. The infected cultures were then plated out on large LB plates (Amp/Tet/Glucose) and allowed to grow at 37°C until the colonies were visible. The colonies were suspended in 2xYT medium and the glycerol cultures were made as above described. This panning round was repeated twice, and in the third round elution was carried out with addition of fluorescein in a concentration of 100 pg/ml in PBS. The enrichment of specific phage antibodies was monitored by panning the initial as well as the subsequent fluorescein-specific sub-libraries against the blocking buffer (Fig. 12). Antibodies with specificity against fluorescein were isolated after 3 rounds of panning.

ELISA measurements One of the criteria for the successful biopanning is the isolation of individual phage clones that bind to the targeted antigen or hapten. We undertook the isolation of anti-FITC phage antibody clones and characterized them first in a phage ELISA format. After the 3rd round of biopanning (see above), 24 phagemid containing clones were used to inoculate 100 pl of 2xYT medium (Amp/Tet/Glucose) in an ELISA plate (Nunc), which was subsequently shaken at 37"C for 5 hrs. 100 p1 of 2xYT medium (Amp/Tet/1 mM IPTG) were added and shaking was continued for minutes. A further 100 pl of 2xYT medium (Amp/Tet) containing the helper phage (1 x 10' cfu/well) was added and shaking was done at RTfor 3 hrs. After addition of kanamycin to select for successful helper phage infection, the shaking was continued overnight. The plates were then centrifuged and the supernatants were pipetted directly into ELISA wells coated with 100 pi FITC-BSA (100pg/ml) and blocked with milk powder. Washing was performed similarly as during the panning procedure and the bound phages were detected with anti-M13 antibody- POD conjugate (Pharmacia) using soluble POD substrate (Boehringer-Mannheim).

Of the 24 clones screened against FITC-BSA, 22 were active in the ELISA (Fig. 13): The initial libraries of similar titer gave rise to no detectable signal.

Specificity for fluorescein was measured in a competitive ELISA. Periplasmic fractions of five FITC specific scFvs were prepared as described above. Western blotting indicated that all clones expressed about the same amount of scFv fragment -34- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 (data not shown). ELISA was performed as described above, but additionally, the periplasmic fractions were incubated 30 min at RT either with buffer (no inhibition), with 10 mg/ml BSA (inhibition with BSA) or with 10 mg/ml fluorescein (inhibition with fluorescein) before adding to the well. Binding scFv fragment was detected using the anti-FLAG antibody M1. The ELISA signal could only be inhibited, when soluble fluorescein was added, indicating binding of the scFvs was specific for fluorescein (Fig. 14).

2.6 Sequence analysis The heavy chain CDR3 region of 20 clones were sequenced in order to estimate the sequence diversity of fluorescein binding antibodies in the library (Fig. 15). In total, 16 of 20 sequences were different, showing that the constructed library contained a highly diverse repertoire of fluorescein binders. The CDR3s showed no particular sequence homology, but contained on average 4 arginine residues. This bias towards arginine in fluorescein binding antibodies had already been described by Barbas et al., 1992.

2.7 Production E. coli JM83 was transformed with phagemid DNA of 3 selected clones and cultured in 0.5 L 2xYT medium. Induction was carried out with 1 mM IPTG at O6 O 0.4 and growth was continued with vigorous shaking at RT overnight.

The cells were harvested and pellets were suspended in PBS buffer and sonicated.

*The supernatants were separated from the cell debris via centrifugation and purified via the BioLogic system (Bio-Rad) by with a POROS®MC 20 column (IMAC, PerSeptive Biosystems, Inc.) coupled with an ion-exchange chromatography column. The ion-exchange column was one of the POROS®HS, CM or HQ or PI (PerSeptive Biosystems, Inc.) depended on the theoretical pl of the scFv being purified. The pH of all the buffers was adjusted to one unit lower or higher than the pl of the scFv being purified throughout. The sample was loaded onto the first IMAC column, washed with 7 column volumes of 20 mM sodium phosphate, 1 M NaCI and mM imidazole. This washing was followed by 7 column volumes of 20 mM sodium phosphate and 10 mM imidazole. Then 3 column volumes of an imidazole gradient (10 to 250 mM) were applied and the eluent was connected directly to the ion-exchanger. Nine column volumes of isocratic washing with 250 mM imidazole was followed by 15 column volumes of 250 mM to 100 mM and 7 column volumes of an imidazole /NaCI gradient (100 to 10 mM imidazole, 0 to 1 M NaCI). The flow rate was 5 ml/min. The purity of scFv fragments was checked by SOS-PAGE Coomassie SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 staining (Fig. 16). The concentration of the fragments was determined fron th3 absorbance at 280 nm using the theoretically determined extinction coefficient (Gill von Hippel, 1989). The scFv fragments could be purified to homogeneity (see Fig. 16). The yield of purified fragments ranged from 5 to 10 mg/L/OD.

Example 3: HuCAL H3K2 Library Against a Collection of Antigens In order to test the library used in Example 2 further, a new selection procedure was carried out using a variety of antigens comprising 3-estradiol, testosterone, Lewis-Y epitope (LeY), interleukin-2 lymphotoxin-13 E-selectin ligand-1 (ESL-1), and BSA.

3.1 Biopanning The library and all procedures were identical to those described in Example 2. The ELISA plates were coated with 3-estradiol-BSA (100 pg/ml), testosterone-BSA (100 pg/ml), LeY-BSA (20 pg/ml) IL-2 (20 pg/ml), ESL-1 (20 pg/ml) and BSA (100 pg/ml), LT-f (denatured protein, 20 pg/ml). In the first two rounds, bound phages were eluted with 0.1 M triethylamine (TEA) at RT for 10 minutes. In the case of BSA, "elution after three rounds of panning was carried out with addition of BSA in a concentration of 100 pg/ml in PBS. In the case of the other antigens, third round elution was done with 0.1 M triethylamine. In all cases except LeY, enrichment of binding phages could be seen (Figure 17). Moreover, a repetition of the biopanning experiment using only the 15-mer library resulted in the enrichment of LeY-binding phages as well (data not shown).

3.2. ELISA measurements Clones binding to 1-estradiol, testosterone, LeY, LT-1, ESL-1 and BSA were further analyzed and characterized as described in Example 2 for FITC. ELISA data for anti- B-estradiol and anti-ESL-1 antibodies are shown in Fig. 18. In one experiment.

selectivity and cross-reactivity of binding scFv fragments were tested. For this purpose, an ELISA plate was coated with FITC, testosterone, 1-estradiol, BSA, and ESL-1, with 5 wells for each antigen arranged in 5 rows, and 5 antibodies, one against each of the antigens, were screened against each of the antigens. Fig. 19 -36- SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 shows the specific binding of the antibodies to the an t igen it was selected for, and the low cross-reactivity with the other four antigens.

3.3 Sequence analysis The sequencing data of several clones against 1-estradiol (34 clones), testosterone (12 clones), LT-1 (23 clones), ESL-1 (34 clones), and BSA (10 clones) are given in Figures 20 to 24.

Example 4: Vector Construction To be able to take advantage of the modularity of the consensus gene repertoire, a vector system had to be constructed which could be used in phage display screening of HuCAL libraries and subsequent optimization procedures. Therefore, all necessary vector elements such as origins of single-stranded or double-stranded replication, promotor/operator, repressor or terminator elements, resistance genes, potential recombination sites, gene III for display on filamentous phages, signal sequences, or detection tags had to be made compatible with the restriction site pattern of the modular consensus genes. Figure 25 shows a schematic representation of the pCAL vector system and the arrangement of vector modules and restriction sites therein. Figure 25a shows a list of all restriction sites which are already incorporated into the consensus genes or the vector elements as part of the modular system or which are not yet present in the whole system. The latter could be used in a later stage for the introduction of or within new modules.

4.1 Vector modules A series of vector modules, was constructed where the restriction sites flanking the gene sub-elements of the HuCAL genes were removed, the vector modules themselves being flanked by unique restriction sites. These modules were constructed either by gene synthesis or by mutagenesis of templates. Mutagenesis was done by add-on PCR, by site-directed mutagenesis (Kunkel et al., 1991) or multisite oligonucleolide-mediated mutagenesis (Sutherland et al., 1995; Perlak, 1990) using a PCR-based assembly method.

-37- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Figure 26 contains a list of the modules constructed. Instead of the terminatoi module M9 (Hindlll-lpp-Pacl), a larger cassette M911 was prepared to introduce Fsel as additional restriction site. M911 can be cloned via Hindlll/BsrGI.

All vector modules were characterized by restriction analysis and sequencing. In the case of module M11-II, sequencing of the module revealed a two-base difference in positions 164/65 compared to the sequence database of the template. These two different bases (CA GC) created an additional Banll site. Since the same twobase difference occurs in the fl origin of other bacteriophages, it can be assumed that the two-base difference was present in the template and not created by mutagenesis during cloning. This Banll site was removed by site-directed mutagenesis, leading to module M11-111. The BssSI site of module M14 could initially not be removed without impact on the function of the ColE1 origin, therefore M14- Ext2 was used for cloning of the first pCAL vector series. Figures 29 to 34 are showing the functional maps and sequences of the modules used for assembly of the modular vector pCAL4 (see below). The functional maps and sequences of additional modules can be found in Figure 35a. Figure 35b contains a list of oligonucleotides and primers used for the synthesis of the modules.

4.2 Cloning vector pMCS To be able to assemble the individual vector modules, a cloning vector pMCS containing a specific multi-cloning site (MCS) was constructed. First, an MCS cassette (Fig. 27) was made by gene synthesis. This cassette contains all those restriction sites in the order necessary for the sequential introduction of all vector modules and can be cloned via the 5'-Hindlll site and a four base overhang at the 3'-end compatible with an Aatll site. The vector pMCS (Figure 28) was constructed by digesting pUC19 with Aatll and Hindlll, isolating the 2174 base pair fragment containing the bla gene and the ColE1 origin, and ligating the MCS cassette.

4.3 Cloning of modular vector pCAL4 This was cloned step by step by restriction digest of pMCS and subsequent ligation i of the modules M1 *(via Aatll/Xbal), M7111 (via EcoRI/Hindlll), and M911 (via Hindlll/BsrGI), and M11-11 (via BsrGI/Nhel). Finally, the bla gene was replaced by the cat gene module M17 (via Aatll/Bglll), and the wild type ColE1 origin by module M14-Ext2 (via Bglll/Nhel). Figure 35 is showing the functional map and the sequence of pCAL4.

-38- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 4.4 Cloning of low-copy number plasmid vectors pCALO A series of low-copy number plasmid vectors was constructed in a similar way using the p15A module M12 instead of the ColE1 module M14-Ext2. Figure 35a is showing the functional maps and sequences of the vectors pCALO1 to pCALO3.

Example 5: Construction of a HuCAL scFv Library 5.1. Cloning of all 49 HuCAL scFv fragments All 49 combinations of the 7 HuCAL-VH and 7 HuCAL-VL consensus genes were assembled as described for the HuCAL VH3-VK2 scFv in Example 2 and inserted into the vector p8S12, a modified version of the pLisc series of antibody expression vectors (Skerra et al., 1991).

5.2 Construction of a CDR cloning cassette For replacement of CDRs, a universal 13-lactamase cloning cassette was constructed having a multi-cloning site at the 5'-end as well as at the 3'-end. The site comprises all restriction sites adjacent to the 5'-end of the HuCAL VH and VL CDRs, the 3'-multi-cloning site comprises all restriction sites adjacent to the 3' end of the HuCAL VH and VL CDRs. Both and 3'-multi-cloning site were prepared as cassettes via add-on PCR using synthetic oligonucleotides as and 3'-primers using wild type 1-lactamase gene as template. Figure 36 shows the functional map and the sequence of the cassette bla-MCS.

5.3. Preparation of VL-CDR3 library cassettes The VL-CDR3 libraries comprising 7 random positions were generated from the PCR fragments using oligonucleotide templates VK1&VK3, Vi2 and VK4 and primers O_K3L_5 and O_K3L_3 (Fig. 37) for the VK genes, and V. and primers (5'-GCAGAAGGCGAACGTCC-3') and O_L3LA_3 (Fig. 38) for the V, genes. Construction of the cassettes was performed as described in Example 2.3.

-39- SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/036 4 7 5.4 Cloning of HuCAL scFv genes with VL-CDR3 libraries Each of the 49 single-chains was subcloned into pCAL4 via Xbal/EcoRI and the VL- COR3 replaced by the 1-lactamase cloning cassette via Bbsl/Mscl, which was then replaced by the corresponding VL-CDR3 library cassette synthesized as described above. This CDR replacement is described in detail in Example 2.3 where the cat gene was used.

Preparation of VH-CDR3 library cassette The VH-CDR3 libraries were designed and synthesized as described in Example 2.3.

5.6 Cloning of HuCAL scFv genes with VL- and VH-CDR3 libraries Each of the 49 single-chain VL-CDR3 libraries was digested with BssHII/Styl to replace VH-CDR3. The "dummy" cassette digested with BssHII/Styl was inserted, and was then replaced by a corresponding VH-CDR3 library cassette synthesized as described above.

Example 6: Expression tests Expression and toxicity studies were performed using the scFv format VH-linker-VL.

All 49 combinations of the 7 HuCAL-VH and 7 HuCAL-VL consensus genes assembled as described in Example 5 were inserted into the vector pBS13, a modified version of the pLisc series of antibody expression vectors (Skerra et al., 1991). A map of this vector is shown in Fig. 39.

E. coli JM83 was transformed 49 times with each of the vectors and stored as glycerol stock. Between 4 and 6 clones were tested simultaneously, always including the clone H3K2, which was used as internal control throughout. As additional control, the McPC603 scFv fragment (Knappik Pluckthun, 1995) in pBS13 was expressed under identical conditions. Two days before the expression test was performed, the clones were cultivated on LB plates containing 30 pg/ml chloramphenicol and 60 mM glucose. Using this plates an 3 ml culture (LB medium SUBSTTUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 containing 90 pg chloramphenicol and 60 mM glucose) was inoc,'lated overnight at 37 Next day the overnight culture was used to inoculate 30 ml LB medium containing chloramphenicol (30 pg/mi). The starting OD 6 oo 0 was adjusted to 0.2 and a growth temperature of 30 °C was used. The physiology of the cells was monitored by measuring every 30 minutes for 8 to 9 hours the optical density at 600 nm. After the culture reached an of 0.5, antibody expression was induced by adding IPTG to a final concentration of 1 mM. A 5 ml aliquot of the culture was removed after 2 h of induction in order to analyze the antibody expression. The cells were lysed and the soluble and insoluble fractions of the crude extract were separated as described in Knappik Pluckthun, 1995. The fractions were assayed by reducing SDS-PAGE with the samples normalized to identical optical densities. After blotting and immunostaining using the a-FLAG antibody M1 as the first antibody (see Ge et al., 1994) and an Fc-specific anti-mouse antiserum conjugated to alkaline phosphatase as the second antibody, the lanes were scanned and the intensities of the bands of the expected size (appr. 30 kDa) were quantified densitometrically and tabulated relative to the control antibody (see Fig. Example 7: Optimization of Fluorescein Binders Construction of L-CDR3 and H-CDR2 library cassettes A L-CDR3 library cassette was prepared from the oligonucleotide template CDR3L (5'-TGGAAGCTGAAGACGTGGGCGTGTATTATTGCCAGCAG(TR5)(TR

I),CCG(TRI)-

TTTGGCCAGGGTACGAAAGTT-3') and primer 5'-AACTTTCGTACCCTGGCC-3' for synthesis of the complementary strand, where (TRI) was a trinucleotide mixture representing all amino acids except Cys, (TR5) comprised a trinucleotide mixture representing the 5 codons for Ala, Arg, His, Ser, and Tyr.

A H-CDR2 library cassette was prepared from the oligonucleotide template CDRsH (5'-AGGGTCTCGAGTGGGTGAGC(TRI)ATT (TRI)(6)(TRI)ACC(TRI)TATGCGGATA *se* GCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCA-3'), and primer 5'-TGGTGTTTTTCGAATTATCA-3' for synthesis of the complementary strand, where (TRI) was a trinucleotide mixture representing all amino acids except Cys, (6) comprised the incorporation of T, resulting in the formation of 6 codons for Ala, Asn, Asp, Gly, Ser, and Thr, and the length distribution being obtained by performing one substoichiometric coupling of the (TRI) mixture during synthesis, omitting the capping step normally used in DNA synthesis.

-41- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 DNA synthesis was performed on a 40 nmole scale, o!igos were oissorl-ed in TP buffer, purified via gel filtration using spin columns (S-200), and the DNA concentration determined by OD measurement at 260 nm (OD 1.0 40 pg/ml).

nmole of the oligonucleotide templates and 12 nmole of the corresponding primers were mixed and annealed at 80°C for 1 min, and slowly cooled down to 37°C within 20 to 30 min. The fill-in reaction was performed for 2 h at 37°C using Klenow polymerase (2.0 pl) and 250 nmole of each dNTP. The excess of dNTPs was removed by gel filtration using Nick-Spin columns (Pharmacia), and the doublestranded DNA digested with Bbsl/Mscl (L-CDR3), or Xhol/Sful (H-CDR2) over night at 37°C. The cassettes were purified via Nick-Spin columns (Pharmacia), the concentration determined by OD measurement, and the cassettes aliquoted pmole) for being stored at 7.2 Library cloning: DNA was prepared from the collection of FITC binding clones obtained in Example 2 (approx. 10' to clones). The collection of scFv fragments was isolated via Xbal/EcoRI digest. The vector pCAL4 (100 fmole, 10 pg) described in Example 4.3 was similarly digested with Xbal/EcoRI, gel-purified and ligated with 300 fmole of the scFv fragment collection over night at 16°C. The ligation mixture was isopropanol precipitated, air-dried, and the pellets were redissolved in 100 p1 of dd H 2 0. The ligation mixture was mixed with 1 ml of freshly prepared electrocompetent SCS 101 cells (for optimization of L-CDR3), or XL1 Blue cells (for optimization of H-CDR2) on ice. One round of electroporation was performed and the transformants were eluted in SOC medium, shaken at 37°C for 30 minutes, and an aliquot plated out on LB plates (AmpfTet/Glucose) at 37°C for 6-9 hrs. The number of transformants was 5 x 104.

Vector DNA (100 pg) was isolated and digested (sequence and restriction map of scH3K2 see Figure 8) with Bbsl/Mscl for optimization of L-CDR3, or Xhol/NspV for optimization of H-CDR2. 10 pg of purified vector fragments (5 pmole) were ligated with 15 pmole of the L-CDR3 or H-CDR2 library cassettes over night at 16°C. The ligation mixtures were isopropanol precipitated, air-dried, and the pellets were redissolved in 100 pl of dd H 2 0. The ligation mixtures were mixed with 1 ml of freshly prepared electrocompetent XL1 Blue cells on ice. Electroporation was performed and the transformants were eluted in SOC medium and shaken- at 37°C for minutes. An aliquot was plated out on LB plates (Amp/Tet/Glucose) at 37°C for 6-9 -42- SUBSTITTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 hrs. The number of transformants (library size) was greater thian -10 tor both libraries. The libraries were stored as glycerol cultures.

7.3. Biopanning This was performed as described for the initial H3K2 H-CDR3 library in Example 2.1.

Optimized scFvs binding to FITO could be characterized and analyzed as described in Example 2.2 and 2.3, and further rounds of optimization could be made if necessary.

C*

C

C C

C

C

*C

C

C

*C*CCC

C

C

CC..

C C

C

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,.:too: SUSiTT SHE RLS6 WO 97/08320 WO 9708320PCT/EP96103647 Table 1A: Human kappa germline gene segments Used Name' Reference' Family' Germline genes' Vk1-I 9 1 08;018;DPKI .Vk-2 1 1 L 14; DPK2 Vkl-3 2 1 Ll 50); HK1OI;HK146; HK189 Vk 1-4 9 1 L11 2 1 Vk1-6 1 1 Vk 1-7 1 1 LFVK431 Vkl1-8 1 1 LI; HK137 VkI-9 1 1 A20;DPK4 VkI-1O 1 1 L1 8; Va" Vkl-l1 1 1 1-4; L18; Va'; V4a Vkl-12 2 1 L5; L190): Vb; VbJ4 DPK5; L19(2); Vb;DPK6 VkI-13 2 1 L15(2); HK134; HK1 66, DPK7 VkI-14 8 1 M8:Vd; DPK8 VklI- 15 8 1 *L9: Ve Vk 1-1,6 1 1 L112(1): HK 102; V1 Vkl-17 2 1 L112(2) **Vk1-18 1 1 0 12a (V3b) *Vk1-19 6 1 02:012;DPK9 *Vkl1-20 2 1 L24; Ve"; V1 3;DPK *Vkl-21 1 1 04; 014 Vkl1-22 2 1 122 Vk1-23 2 1 L23 Vk2-1 1 2 A2; DPK 12 Vk2-2 6 2 01:;011(1); DPK13 Vk2-3 6 2 0O12(2); V3a Vk2-4 2 2 L13 1 2 DPK14 Vk2-6 4 2 A3, A19; Vk2-7 4 2 A29; DPK27 Vk2-8 4 2 A13 Vk2-9 1 2 A23 SUBSTIUT SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 1A: (continued) Used Name' Reference' Family' Germline genes' Vk2- 10 4 2 A7; DPK 17 Vk2-1 1 4 2 All7; DPKI 8 Vk2-1 2 4 2 All;DPK19 Vk3-1 11 3 All1; humkv3O5; Vk3-2 1 3 L20; Vg" Vk3-3 2 3 L2; L16; humkv328; humkv328h2; humkv328h5; DPK2l Vk3-4 I11 3 A27; humkv325;VkRF;0DPK22 2 3 L25; DPK23 Vk3-6 2 3 L 10(1) Vk3-7 7 3 L 10(2) Vk3-8 7 3 L6; Vg Vk4-1 3 4 3:VkIV; DPK24 Vk5-1 105 12; Vk6-1 12 6 A14; Vk6-2 12 6 AlO; A26; DPK26 Vk7-1 5 7 Bi .4 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCTI/EP96/03647 Table 1 B: Human lambda germline gene segments Used Name, DPL1 DPI-2 o P13 DPL4 HUMLV1 17 o PL5

DPLG

O PL7 0 PL8 DPL9 DPLI 0 VIAMBDA 2.1 DPLI 1 DPL1 2 DPIL13 DPI.14 DPLI16 DPL23 Humlv318 DPL18 DPI 19 DPL21 HUM IV801 oDPL22 0PL24 gVLX-4.4 Reference' Family' Germline genes' 1 HUMIVi Li 1 HUMLV122 1 VLAMBDA 1.1 1 HUMLV1 17D 1 IGLVIS2 1 HUMLVIO42 I HUML-Vii 2 2 2 2 2 2 3 Humlv418; IGI -3 VI 111.1 3 7 4A; HUMIGIVA 7 8 V18.1 8 9 unassigned VLAMBDA N.2 V3S 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCr/EP96/03647 Table 1iC: Human heavy chain germline gene segments Used Name' Reference, Family' Germline genes 4 o 0 VH 1-12 -1 VH1-12-8 VHI-12-2 VHI-12-9 VHI-12-3 VH1-12-4 VH1-12-5 VHI-12-6 VHI-12-7 VH1-13-1 VH I- 13-2 VH i- 13-3 VH 1 -13-4 VHI-13-5 VHI-13-6 VHI-13-7 VHI-13-8 VH1-1:3-9 VH1-13-10 VH1-13-1 I VHI-13-12 VH1-13-13 VH1-13-14 VH1-13-l'S VH1-13-16 VH 1-13-17 VH1-13-18 VH1-13-19 VH 1-1X- 1 VH2 -21 -1 VH2-31-1 VH2-31-2 VH2-31-3 VH2-31-4 VH 2-31-5 VH2-31-6 DP 10; DA- 2; DA- 6 RR.VH 1.2 hv1263 YAC-7; RR.VH 1.1; 1-69 DP3 0P21I; 4d27 5a; VH 7a 1-4.l1b; V/1-4.1 b 10D37; VH7b ;7-8 1; YAC- DP14; VH IGRR; V I-18 71-5; DP2 E3- DPi V3 VI-2b 1-2; VI-2 DP8 1-1 DP1 2 V13C 1-3b; DP25; Vl-3b 1-92 1-3; V1 -3 21-2; 3- 1: DP7; V 1-46 HG3 DP4; 7-2; Vi1-45 COS DP5: 1-24P 11-5b VH2S12-l VH2S12-7 VH2S12-9; DP27 VHS 12-10 V12-26; DP2G; 2-26 VF2 -26 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table I C: (continued) Used Name, VH2-31-7 VH2-31-14 VH2-31-8 VH2-3 1-9 VH2-3 1 -10 VH2-3 1 -11 VH2-31-12 VH2-31-13 VH3-1 1 -1 VH3-1 1-2 VH3-i 1-3 VH3-1 1-4 \/H3-i 1-5 VH3-1 1 -6 VH3-1 1-7 VH3-i 1-8 VH3-13-1 VH3-i 3-5 VH3-13-6 VH3-13-7 VH3-13-8 VH3-1 3-9 VH3-13-10 VH3-13-1 1 VH3-13-12 VH3-13-13 VH3-13-14 VH3-13-1 5 VH3-13-16 VH3-13-17 VH3-13-18 VH3- 13-19 VH3-13-20 VH3-13-21 VH-3-13-22 VH3- 13-23 Reference' Family' Germline genes' 0 0 0* 0 19 7 2 2 18 2 2 2 13 19 3 19 14 19 3 14 3 19 25 19 24 5 19 3 19 3 24 27 19 16 19 19 14 14 14 14 DP28; DA-7 YAC-3; 2-70 VH2Si VH2S12-12 11-5; VH2Sl 2-2; VH2SI2-8 VH2S1 2-4: VH2S1 2-6 VH2Sl2-14 v65-2; DP44 D 13-2; DP48 DP52 v3- 13 DP42 8-18; YAC-5; 3-G6 V3-53 22-28; DP3S; V3-1 1 DP59; VH 19; V3-35 f I-pi; DPG I DP46; GL-SJ2; COS 8; hv3005: hv3005f3; 3d21 b; 5 6 p1I VH 26 vh2fic DP47; VH26; 3-23 1-91 DP58 1-9111, DP49; 3-30; 3d28.1 3019139; DP50; 3-33; 3d277 COS 3 DP5 1 HI I DP53: COS 6; 3-74; D A-8 DP54; VH3-1 1; V3-7 V3-64; YAC-6 V3-48 V3-43; DP33 V3-33 SUBSTITUJTE SHEET(RULE 26) WO 97/08320 PTE9/34 PCT/EP96/03647 Table IC: (continued) Used Name' Reference' Family' Germline genesA VH3- 13-24 VH3-.13-25 VH3-1 3-26 VH3-14-1 VH3-14-4 VH3-14-2 VHJ-14-3 VH3- IX- 1 VH3-1X-2 VH3-1X-3 VH3-IX-4 VH3-1X-5 VH3-1X-6 VH3-1X-7 VH3-1X-8 VH3-1X-9 VH4-1 1 -1 VH4-1 1-2 VH4-1 1-3 VH4-1 1-4 VH4-1 1-5 VH4-11-6 VH4-1 1-7 VH4-1 1 -8 VH4-1 1-9 VH4-1 1-10 VH4-1 1-11 VH4-1 1-12 VH4-1 1-13 VH4-1 1-14 VH4-1 1-IS VH4-11-16 VH4-2 1-1 VH4 -2 1-2 VH4-21-3 V3-21; DP77 V3-20; DP32 V3-9; DP31 12-2; DP29; 3-72; DA-3 YAC-9; 3-73; MTGL VHD26 LSG8.1; LSG9.1; LSG1o.i; HUM121GVH; HUM131GVH ISGi 1.1: HUM41GVH 9-1; DP38; LSG7.1; RCG1.1: LSG1.1; LSG3.1: LSG5.1; HUM I 5IGVH: HUM2iGVH; HUM91GVH LSG4.1 LSG2.1 LSG6.1; HUM iOIGVH.

3-15; V3-15 LSG12.1; V3-49 Tou-VH4.2 1 VH4.21; DP63; VHS; 4076; V4-34 4.44 4.44.3 4.36 4.37 IV-4; 4.35; V4-4 VH4.1 1; 3d 197d; DP71; 5 8 p2 H7 H8 H9 VH4.16 4.38 VH4.15 58 71-4; V4-59 VH4.17: VH4.23; 4d255; 4.40; DP69 VH4.19; 79; V4-4b SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table I C: (continued) Used Name' VH-4-21-4 VH4-21-5 VH-4-21 -6 VH-4-21-7 VH-4-2 1-8 VH-4-21-9 VH4-31 -1 VH-4 -3 1-2 VH-4-31-3 VH-4 -3 1-4 VH-4-31 -5 VH-4-31-6 VH-4-31-7 VH,4-31 -8 V114-31-9 VH-4-31-1O VH-4-3 1-1 1 VH-4-31-12 VH4-31-13 VH-4-31-14 'JH4-31-15 VH-4-31-16 VH-4-31-17 VH-4-31-18 VH4-31-19 VH-4-3 1-20 VHS1-12-1 VH-5-12-2 VHS1-12-3 VHS1-12-4 VH-6-35- 1 Reference' Family 3 Germline geneS' DP7O; 4d68; 4.41 DP67; VH4-4B VH-4.22; VHSP; VH-JA VH4.13; 1-911; 12G- I 3d28d; 4.42; DP68; 4-28 hv4005; 3d24d VH4.14 4.34; 3d230d; DP78 4.34.2 DP64; 3d2l6d DP65; 4-3 1; 3d277d 4.33; 3d75d HIl 4.31 4.32 3d27 7d 3d216d 3d279d VH-4.18: 4d 154; DP79 V4-39 2-1; DP79 4.30 VH-4.12 7 1-2; DP66 4.39 V4-61 VH251: DP73; VHVCW; 51 -RI; VHVLB; VHVCH; VHVT; VHVAU, VHVBLK: VhAU:

VHVJB

I1-v: DP8O; 5-78 VH32; VHVRG; VHVMW; 5-21 VHVI: VH-6; VHVIIS; VHVITE;, VHVIJB; VHVICH; VHVICW; VHVIBLKVHVIMW; DP74; 6-]I;VG-i 59.

SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2A: rearranged human kappa sequences Name' aa' Computed Germline Diff. to diff. to Reference family 3 gene" germlines germline 6 Ill-3R 108 1 08 1 1,1% No.86 109 1 08 3 3,2% AU 108 1 08 6 6,30% 103 ROY 108 1 08 6 6,3% 43 IC4 108 1 08 6 6.3% HIV-826 106 1 08 3 3,2% 8 GRI 108 1 08 8 8,4% AG 106 1 08 8 8,6% 116 REI 108 1 08 9 9,5% 86 CLL PATIENT 16 88 1 08 2 2,3% 122 CLL PATIENT 14 87 1 08 2 2,30/0 122 CLL PATIENT 15 88 1 08 2 2,3 0 /a 122 GM4672 108 1 08 11 11,6% 24 HUM. YFC 51.1 108 1 08 12 12,6%b 110 LAY 108 1 08 12 12,6 0 48 HIV-b13 106 1 08 9 9,7% 8 MAL-NaCI 108 1 08 13 13,7% 102 STRAb SA-1A 108 1 02 0 0,0% 120 HuVHCAMP 108 1 08 13 13,7 0 o 100 .CRO 108 1 02 10 10,5% SAml07 108 1 02 12 1 2,6 0 /o 108 WALKER 107 1 02 4 4,2% 57 1Il-2R 109 1 A20 0 FOG1-A4 107 1 A20 4 4,2% 41 HK137 95 1 L1 0 00%/ CEA4-8A 107 1 02 7 7,4 0 41 Va' 95 1 L4 0 0,00/o TR1.21 108 1 02 4 4.2 0 92 HAU 108 1 02 6 6,3% 123 SHK102 95 1 L12(1) 0 O,0/a 9 H20C3K 108 1 L12(2) 3 3,2 0 125 CHEB 108 1 02 7 7 HK134 95 1 L15S(2) 0 TEL9 108 1 02 9 9 ,5 0 73 Z3 SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03 6 4 7 Table 2A: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' family' gene 4 germiine' germlineG TR1.32 103 1 02 3 3,2% 92 RF-KESI 97 1 A20 4 4,2% 121 WES 108 1 L5 10 lo.% 61 Dllpi 95 1 04 1 1,1/ SA-4B 107 1 112(2) 8 8,4% 120 HK1OI 95 1 115(1) 0 o,0% 9 TR1.23 108 1 02 5 5,3% 92 HF2-1/17 108 1 A30 0 0,0o/ 4 2E7 108 1 A30 1 1,1%/a 62 33.C9 107 1 12(2) 7 7 ,4 0 126 3D6 105 1 112(2) 2 2 ,1/o 34 1-2a 108 1 L8 8 8,4% RF-KI 97 1 8 4 4 121 TNF-E7 108 1 A30 9 9 41 TR1.22 108 1 02 7 7,4/a 92 HIV-835 106 1 02 2 2 ,2a 8 HIV-b22 106 1 02 2 2 8 HIV-b27 106 1 02 2 2 8 HIV-B8 107 1 02 10 lo8'o 8 HIV-b8 107 1 02 10 10,8/ 8 RF-SJ 95 1 A30 5 5,3% 113 GAL(l) 108 1 A30 6 6,30/ 64 R3.SHSG 108 1 02 6 6.3% HIV-b14 106 1 A20 2 2.2% 8 TNF-EI 105 1 .5 8 8,4/o 41 WEA 108 1 A30 8 8 ,4 0 37 EU 108 1 L12(2) 5 5,3 0 FOGI-G8 108 1 18 11 11,6% 41 1X7RGi 108 1 1 8 8 BLI 108 1 18 3 3,2% 72 SKUE 108 1 L12(2) 11 11,6% 32 LUNmO 1 108 1 L12(2) 10 10,5% 6 HIV-bl 106 1 A20 4 4 8 HIV-s4 103 1 02 2 2.2% 8 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCTIEP96/03647 Table 2A: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' family 3 gene' germline' germline' CAR 107 1 L12(2) 11 11,7% 79 BR 107 1 112(2) 11 11.6% CLL PATIENT 10 88 1 02 0 122 CLL PATIENT 12 88 1 02 0 122 KING 108 1 L12(2) 12 12.60%0 V13 95 1 L24 0 46 CLL PATIENT 11 87 1 02 0 122 CLL PATIENT 13 87 1 02 0 0,0%o 122 CLL PATIENT 9 88 1 012 1 1,1 122 HIV-B2 106 1 A20 9 9,7% 8 HIV-b2 106 1 A20 9 9,7% 8 CL PATIENT 5 88 1 A20 1 1,1% 122 CL PATIENT 1 88 1 L8 2 2.3% 122 CLL PATIENT 2 88 1 L8 0 122 CLL PATIENT 7 88 1 LS 0 122 CLL PATIENT 8 88 1 L5 0 0,00/o 122 HIV-bS 105 1 L5 11 12,0 0 /o 8 .CLL PATIENT 3 87 1 18 1 1,1% 122 SCLL PATIENT 4 88 1 L9 0 0.0%o 122 CLL PATIENT 18 85 1 L9 6 7,10/% 122 CLL PATIENT 17 86 1 L12(2) 7 122 HIV-b20 107 3 A27 11 11,7% 8 2C12 108 1 L12(2) 20 21,10/% 68 1811 108 1 L12(2) 20 21,1% 68 1H1 108 1 L12(2) 21 22,1% 68 2A12 108 1 L12(2) 21 22,1010 68 **CUR 109 3 A27 0 66 GLO 109 3 A27 0 16 RF-TS1 96 3 A27 0 121 GAR' 109 3 A27 0 67 FLO 109 3 A27 .0 66 PIE 109 3 A27 0 91 HAH 14.1 109 3 A27 1 1,0% 51 HAH 14.2 109 3 A27 1 1,0% 51 SUBSTITUTE SHEET(RULE 26) WO 97/08320 PCI/EP96/03647 Table 2A: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' family 3 gene 4 germline' germline' HAH 16.1 109 3 A27 1 1,0% 51 NOV 109 3 A27 1 1,0% 52 33.F12 108 3 A27 1 126 8E10 110 3 A27 1 1,0% TH3 109 3 A27 1 1,0%f HIC 108 3 A27 0 0,0% 51 SON 110 3 A27 1 1.0%o 67 PAY 109 3 A27 1 1,0% 66 GOT 109 3 A27 1 1,0% 67 mAbA6H4C5 109 3 A27 1 1,0% 12 BOR' 109 3 A27 2 2,1% 84 RF-SJ3 96 3 A27 2 2,1 0 121 SIE 109 3 A27 2 2,1 0 ESC 109 3 A27 2 2,1% 98 HEW' 110 3 A27 2 2,1% 98 YES8c 109 3 A27 3 3,1 0 33 TI 109 3 A27 3 3,1010 114 mAb 113 109 3 A27 3 3,1 0 71 HEW 107 3 A27 0 94 BRO 106 3 A27 0 0,0 0 /a 94 ROB 106 3 A27 0 94 NG9 96 3 A27 4 4,2% 11 NEU 109 3 A27 4 4.2 0 66 WOL 109 3 A27 4 4,2% 2 3SG6 109 3 A27 4 59 RF-SJ4 109 3 All 0 0,a/0% 88 KAS 109 3 A27 4 4,2% 84 BRA 106 3 A27 1 1,1 94 HAH 106 3 A27 1 1 94 HIC 105 3 A27 0 94 FS-2 109 3 A27 6 6 ,3 0 87 JH' 107 3 A27 6 6, 30 38 EV1-15 109 3 A27 6 6 83 SCA 108 3 A27 6 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCIP606' PCT/EP96103647 Table 2A: (continued) Name' aaz Computed Germline Diff. to diff. to Reference' family' gene' germline 5 germline' mAbl 12 109 3 A27 6 71 sic 103 3 A27 3 94 SA-4A 109 3 A27 6 120 SER 108 3 A27 6 6,30/ 98 GOL' 109 3 A27 7 82 BSGIOK 105 3 A27 9 125 HG2B10K 110 3 A27 9 125 Taykv322 105 3 A27 5 5,4 0 /o 52 CILPATIENT 24 89 3 A27 1 1,10/ 122 HIV-b24 107 3 A27 7 8 HIV-b6 107 3 A27 7 8 Taykv31O 99 3 A27 1 1,10/ S2 KA3D)1 108 3 16 0 0,0 0 191E7 107 3 L6 0 .0,0 0 126 rsvGL 109 3 A27 12 12,5 0 7 Taykv32O 98 3 A27 1 1,2 0 /6 52 Vh 96 3 L 10(2) 0 0,0 0 /0 89 158 108 3 L6 1 1,1/o 109 151 108 3 16 1 109 L S2S3-3 107 3 L6 2 2,10/ 99 152 108 3 16 1. 1,1 0 109 LS7 108 3 L6 1 1,1 0 109 LS2S3-4d 107 3 16 2 2,1 0 99 *LS2S3-4a 107 3 16 2 99 LS4 108 3 16 1 109 156 108 3 16 1 1,10/ 109 *LS2S3-10a 107 3 16 2 99 L52S3-8c 107 3 16 2 99 155 108 3 L6 1 1,1 0 109 LS2S3-5 107 3 16 3 3,20/ 99 *LUNmO3 109 3 A27 13 13.5%/ 6 *IARC/B141 108 3 A27 13 13,7 0 sIkv22 99 3 A27 3 3,50t 13 POP 108 3 16 4 4,20/a ill SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/0364 7 Table 2A: (continued) Name' aa 2 Computed Germline- Diff. to 0/ diff. to Reference' family, gene" germline' germline' 152 53-l0b LS2S3-8f LS2S3-12 HIV-1330 HIV-B320 HIV-b3 HIV-s6

YSE

POM

Humkv328

CLL

LES

HIV-S7 slkvlI Humka3les sIkv12 RF-TS2 11-1 HIV-s3 RF-TMC 1

GER

GF4/1.1 mAbi 114 HIV-10OP13 bkv,6 CLL PATIENT 29 slkv9 bkv17 slkv14 slkv IG bkv3 bkv6 107 3 107 3 107 3 107 3 107 3 108 3 104 3 107 3 109 3 95 3 109 3 96 3 104 3 104 3 99 3 95 3 101 3 95 3 109 3 105 3 96 3 109 3 109 3 109 3 109 3 86 3 86 3 98 3 99 3 99 3 101 3 101 3 99 3 100 3 L6 L6 L6 A2 7 A2 7 A2 7 A27 12/1-16 12/1-i1 12/116 12/1-16 A2 7 A2 7 A2 7 12/1-16 12/116 A2 7 L6 12/116 12/116 1 6 L16 16 16 16 L6 16 L6 3,20/ 3,2%/ 3,20/ 11,7%/ 11,70/ 1 1,7 0 9.9%/ 1,10/ 9 .4 0 1,10/0 3,2%/ 3 ,2 0 12,1%Ol 12,1%/ 8,10/ 4.2%/ 9 ,2 0 3,20/ 4.2%/ 1 4,3 0 10,5%/ 7,4%/ 8,40/.

6,3 0 b 7,4%I 1.2%/ 1,2%/ 3,5%/ 1,2%/ 1.2%/ 2,30/ 4,70/ 2. 3%/ 3,5%/ 99 99 99 8 8 8 8 72 53 19 47 38 8 8 13 18 13 121 8 121 36 71 8 13 122 13 13 13 13 13 13 13 SUBS111TUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT1EP96103647 Table 2A: (continued) Name' aa 2 Computed Germline Diff. to 0/0 d iff. to Reference' family' gene' germline' germline 6 R6B8K 108 3 12/116 12 12,6%/ 125 AL 700 107 3 12/116 9 9,50/ 117 sIkvll1 100 3 12/1-16 3 13 slkv4 97 3 16 4 4,8%1 13 CILL PATIENT 26 87 3 12/116 1 122 AL Se124 103 3 11L16 9 117 slkvl3 100 3 L2/L 16 6 7,0% 13 bkv7 100 3 12/116 5 13 bkv22 100 3 L2/116 6 13 CLL PATIENT 27 84 3 12116 0 122 100 3 16 8 13 CLL PATIENT 25 87 3 12/1 16 4 4,60/ 122 slkv3 86 3 12/116 7 8,10/ 13 slkv7 99 1 02 7 13 HuFd79 ill 3 12(1 16 24 24.2%/ 21 RAD 99 3 A27 9 10,3%1 78 CILL PATIENT 28 83 3 12/116 4 122 REE 104 3 12/116 25 27,2%/ :**FR4 99 3 A27 8 77 .*MD3.3 92 3 16 1 54 M03.1 92 3 16 0 0.0%0 54 GA3.6 92 3 16 2 2,6 0 /a 54 92 3 16 3 3,80/ 54 *WEI* 82 3 A27 0 0,00I0 MD3.4 92 3 L2/116 1 54 MD3.2 91 3 16 3 3,8 0 54 *VER 97 3 A27 19 22,40/ CLL PATIENT 30 78 3 16 3 122 M3.1 N 92 3 12/11 6 1 54 MD3.6 91 3' 12/1 16 0 54 O O M D3 8 91 3 12/116 0 0,0% 54 GA3.4 92 3 16 7 9,00/0 54 M3.6N 92 3 A27 0 54 MD3.1O 92 3 A27 0 54 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2A: (con tinued) Name' aaz Computed Germline Diff. to %f diff. to Reference' family 3 gene' germline' germlinel MD3.13 91 3 A27 0 0,0 0 54 M03.7 93 3 A27 0 0,0 0 54 MD3.9 93 3 A27 0 0,0/ 54 GA3.1 93 3 A27 6 54 bkv32 101 3 A27 5 13 93 3 A27 5 54 GA3.7 92 3 A27 7 54 MD3.12 92 3 A27 2 54 M3.2N 90 3 L 6 6 7,80/ 54 92 3 A27 1 54 WA.N 91 3 1-2/116 8 10 54 M3.8N 91 3 1-2/1.16 7 54 M3.7N 92 3 A27 3 54 GA3.2 92 3 A27 9 11.4%/ 54 GA3.8 93 3 A27 4 54 GA3.3 92 3 A27 8 10.1%/ 54 M3.3N 92 3 A27 5 54 B6 83 3 A27 8 11,3%1 78 E29.1 KAPPA 78 3 1-2/116 0 0,0 0 22 082 12 31 REI-based CAMPATH-9 107 1 08 14 14,70/o 39 RZ 107 1 08 14 14.7% B1 108 1 08 14 14,7% 14 AND 107 1 02 13 13,7% 69 *2A4 109 1 02 12 12,6%/ 23 KA 108 1 08 19 20 107 MEV 109 1 02 14 14 29 DEE 106 1 02 13 1 4 76 OU(IOC) 108 1 02 18 18.90/ HuRSV19VK ill 1 08 21 21 115 SP2 108 1 02 17 17,9%/ 93 BJ26 99 1 08 21 24,1%/ 1 NI1 112 1 08 24 2 106 BMA 03 1OEUCIV2 106 1 L12(i) 21 2 105 SU1BSTITUTE SHEET (RUJLE 26) WO 97/08320 PCT/EP96/03647 Table 2A: (continued) Name' aa' Computed Germline Diff. to 0/ diff. to Reference' family' gene' 9ermline5 germline' CLL PATIENT 6 71 1 A20 0 0,0 0 /o 122 BJ19 85 1 08 16 21.9% 1 GM 607 113 2 A3 0 0.0% 58 R5A3K 114 2 A3 1 1,0% 125 R1C8K 114 2 A3 1 1,0% 125 VK2.R149 113 2 A3 2 2,0 0 118 TR1.6 109 2 A3 4 4,0 0 92 TR1.37 104 2 A3 5 5,0 0 92 FS-1 113 2 A3 6 6,00/% 87 TR1.8 110 2 A3 6 6,00/0 92 NIM 113 2 A3 8 8.00/% 28 Inc 112 2 A3 11 11,0% TEW 107 2 A3 6 96 CUM 114 2 01 7 6,9% 44 HRF1 71 2 A3 4 5,6% 124 CLL PATIENT 19 87 2 A3 0 122 CLL PATIENT 20 87 2 A3 0 0,0 0 /a 122 MIL 112 2 A3 16 16,20/% 26 FR 113 2 A3 20 20,0%/ 101 MAL-Urine 83 1 02 6 8,6% 102 Taykv306 73 3 A27 1 1,6% 52 Taykv312 75 3 A27 1 1,60/0 52 HIV-b29 93 3 A27 14 17,5% 8 1-185-37 110 3 A27 0 0,0 0 /a 119 1-187-29 110 3 A27 0 0,0 0 /a 119 TT117 110 3 A27 9 9,4% 63 HIV-loop8 108 3 A27 16 16,80/% 8 rsv23L 108 3 A27 16 16,8% 7 HIV-b7 107 3 A27 14 1 4,9% 8 ***HIV-bi 107 3 A27 15 16.0% 8 ***HIV-LCi 107 3 A27 19 20,2 0 8 HIV-LC7 107 3 A27 20 21,3% 8 HIV-LC22 107 3 A27 21 2 2,3 0 8 HIV-LC13 107 3 A27 21 22,3% 8 a-, SUBSTTUTE SHEET(RULE 26) WO 97/08320 PCT/EP96/03647 Table 2A: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' family' gene 4 germline' germline' HIV-LC3 107 3 A27 21 22,3%/ 8 107 3 A27 21 22,3% 8 HIV-LC28 107 3 A27 21 22,3% 8 HIV-b4 107 3 A27 22 23,4% 8 CLL PATIENT 31 87 3 A27 15 17,2% 122 HIV-loop2 108 3 L2/L16 17 17,9% 8 108 3 L2/1 16 17 17,9% 8 HIV-LC11 107 3 A27 23 24,5% 8 HIV-LC24 107 3 A27 23 24,5/o 8 HIV-bl2 107 3 A27 24 25.5% 8 107 3 A27 24 25,5% 8 HIV-b21 107 3 A27 24 25,5% 8 HIV-LC26 107 3 A27 26 27,7% 8 G3D10K 108 1 L12(2) 12 12,6% 125 TT125 108 1 L5 8 8,4% 63 HIV-s2 103 3 A27 28 31.1% 8 265-695 108 1 L5 7 7,4% 3 2-115-19 108 1 A30 2 2,10% 119 rsv13L 107 1 02 20 21,1 0 7 HIV-bl8 106 1 02 14 15,1 0 8 RF-KL5 98 3 L6 36 36,7% 97 ZM1-1 113 2 A17 7 7,0 0 3 HIV-s8 103 1 08 16 17.8% 8 K- EViS 95 5 82 0 112 RF-TS3 100 2 A23 0 0,0 0 121 HF-21/28 111 2 A17 1 1,0 0 17 RPMI6410 113 2 A17 1 1,0% 42 JC 1i 113 2 A17 1 1.0% 49 0-81 114 2 A17 5 5,0 0 FK-001 113 4 83 0 0,0% 81 CD5+.28 101 4 83 1 1,0% 27 LEN 114 4 83 1 1,0% 104 UC 114 4 83 1 1,0% 111 101 4 83 1 1,0% 27 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96103647 Table 2A: (continued) Name' aa' Computcd Germline Diff. to diff. to Reference' family' gene" germlinc 5 germline" CD5+.26 101 4 B3 1 27 CD5+.12 101 4 B3 2 27 CD5+.23 101 4 B3 2 27 CD5+.7 101 4 B3 2 27 Vii 113 4 B3 3 56 LOC 113 4 B3 3 72 MAL 113 4 B3 3 72 CDS+.G 101 4 B3 3 27 H2F 113 4 B3 3 PB171V 114 4 B3 4 74 CD5+.27 101 4 B3 4 27 C05i.9 101 4 B3 4 27 CD5-.28 101 4 B3 5 5,00/ 27 CDS-.26 101 4 B3 6 5.9%I 27 CD5+.24 101 4 B3 6 27 CD5+.10 101 4 B3 6 27 CDs-.i9 101 4 B3 6 27 *CD5-.18 101 4 B3 7 27 *.CID 5-.16 101 4 83 8 27 *CDS-.24 101 4 B3 8 .27 *CDS-.1 7 101 4 B3 10 9,9 0 27 MD4.1 92 4 B3 0 54 MD4.4 92 4 83 0 0,00/ 54 92 4 83 0 54 *MD4.6 92 4 B3 0 0,0 0 54 MD4.7 92 4 B3 0 0,0 0 54 MD4.2 92 4 E13 1 1,3 0 54 *MD4.3 92 4 B3 5 54 CILL PATIENT 22 87 2 A17 2 2 122 CLL PATIENT 23 84 2 A17 2 2 122 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2B3: rearranged human lambda sequences Name' Ra Computed Germline Diff. to diff. to Reference' family' gene' germlne' germlineG WAH 110 1 DPL3 7 68 IB91F2 112 1 DPL3 7 9 DIA 112 1 DPL-2 7 36 mAb67 89 1 DPL3 0 00% 29 HiH-2 110 1 DPL3 12 11/0 3 NIG-77 112 1 DPL-2 9 90/0 72 OKA 112 1 DPL-2 7 84 KOL 112 1 DPL-2 12 11%/ III 1 DPL5 0 6 T2:C 14 110 1 DPLS5 0 6 PR-TS 1 110 1 DPL5 0 00/ 4G12 III 1 DPI5 1 10/% KIM46L 112 1 HUMI..VI17 0 8 Fog-B III 1 DPL-5 3 3 0 31 9F2L III 1 DPL5 3 79 mAbI Il 110 1 DPL5 3 3 0 48 PHOXi 1511l I DPL5 4 4% 49 B12 11 1DPL5 4 4% 74 NIG-64 11 1DPLS 4 4% 72 RF-5SJ2 100 1 DPL-5 6 78 AL EZI 112 1 DPL5 7 41 ZIM 112 1 HUMLV17 7 7 0 18 RF-SJ 1 100 1 DPL5 9 78 IGLV1.1 98 1 DPL4 0 1 *NEW 112 1 HUMLVIi17 1 1 10%/ 42 CB-201 87 1 DPL-2 I 1I/0 62 0 .0:MEM 109 1 OPL.2 6 H210 Il 2 DPL10 4 NOV 110 2 DPL10 8 80/ NEI ill 2 DPL1O 8 80/ 24 AL MC 110 2 OPI 1 6 6 0 /h 28 MES 112 2 DPLI 1 8 80/ 84 FOG I-A3 Ill 2 DPLi 1 9 27 AL NOV 112 2 OPLII 7 28 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2B: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' family' gene' germline' germline' HMST- 1 110 2 DPL1I 4 82 HBW4-I 108 2 DPL12 9 9 0 /0 52 WH 110 2 DPL11I I11 h1/% 34 11-50 1 10 2 DPL1 1 7 82 HBp2 110 2 DPL12 8 3 NIG-84 113 2 OPLil 112 11%/ 73 VIL 112 2 DPL1 1 9 901 58 TRO Ill 2 DPL12 10 10%/ 61 ES492 108 2 DPLI 1 15 15%/ 76 mAb216 89 2 DPL12 1 1% 7 BSA3 109 3 DPLI6 0 0%1 49 THY-29 110 3 OPLiG 0 27 PR-TS2 108 3 DPL16 0 E29.1 LAMBDA 107 3 DPL16 1 13 mAb63 109 3 DPL16 2 29 TEL 14 110 3 DPL16 6 60/ 49 6H-3C4 108 3 DPL16 7 39 SH 109 3 DPLI6 7 .AL GL 109 3 DPLI6 8 80/ 23 H6-3C4 108 3 DPL16 8 83 *V-lambda-2.DS I11 2 DPLI 1 3 30/ 8.12 ID 110 2 DPLI 1 3 81 DSC I11 2 DPL1 1 3 3% 56 PVI 1 110 2 DPLI 1 1 10/ 56 *o 33.H 11 110 2 DPLI 1 4 40% 81 AS1 7 Ill 2 DPL1 1 7 56 900:SD6 110 2 OPLll1 7 70/ 56 0 KS3 110 2 DPLI 1 9 90/ 56 PV6 110 2 DPL12 S S5% 56 NGD9 110 2 DPLI 1 7 70/ 56 MUdi-I 11 2 DPLI 1 11 100/ 27 A3Oc 11 2 DPL1O 6 60/ 56 KS6 110 2 DPLI2 6 6 0 56 TEL13 I1i 2 DPLl I 11 10 0 49 SUBSTITUTE SHEET (RULE 26i) WO 97/08320 WO 9708320PCr/EP96/03647 Table 2B3: (continued) Name' aa' Computed Germline Di ff. to diff. to Reference' family' gene' germline' germline' AS7 110 2 DPL1 2 6 6 0 56 MCG 112 2 DPL112 12 110/a U266L 110 2 DPLI 2 13 1 2 0 /a 77 PR-SJ2 110 2 DPI2 14 13%/ BOH 112 2 DPI2 I11 100/a 37 TOG 111 2 DPLI 1 19 18%/ 53 TEL16 ill 2 OPL1 1 19 18 0 /0 49 No.13 110 2 DPLIO 14 13%/ 52 BO 112 2 DPL12 18 17%/ WIN 112 2 DPL12 17. 16%/ 11 BUR 104 2 DPL12 15 15%/ 46 NIG-58 110 2 DPL12 20 19 0 69' WEIR 112 2 DPLIi1 26 25/ 21 THY-32 Ill 1 DPL8 8 80o0 27 TNF-H9G1 Ill I DPL8 9 90/a 27 mAb6i I1 1l DPL-3 1 1O/% 29 LIiLl 98 1 DPL2 0 54 HA 113 1 DPL3 14 13%/ 63 *LAilll Ill 1 DPL2 3 54 RHE 112 1 OPLi1 17 16 0 16 22 *KIB12L 113 1 DPL8 17 16%/ 79 LOC 113 1 DPL2 15 14%/ 84 NIG-Si 112 1 DPL-2 12 11%/ 67 NEWM 104 1 DPL8 23 22%/ MD3-4 106 3 DPL23 14 13 0 4 Cox 112 1 DPL-2 13 12%/ 84 HiHiG 106 3 DPL23 13 12%/ 3 VOR 112 1 DPL-2 16 isa/ 16 AL POL 113 1 DPL-2 16 15%/ 57 CD4-74 Ill I DPL2 19 18%/ 27 *AMYLOID MOL 102 3 DPL23 15 150/ OS 0T577 108 3 Humlv3l8 10 10 0 4 NIG48 113 1 DPL-3 42 4 66 CARR 108 3 DPL23 18 170/ 19 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2B: (continued) Name' aa' Computed Germline Diff. to diff. to Reference' Sfamily 3 gene germline' germline 6 108 3 DPL23 14 13% 29 NIG-68 99 3 DPL23 25 26% 32 KERN 107 3 DPL23 26 25% 59 ANT 106 3 DPL23 17 16% 19 LEE 110 3 DPL23 18 17% CLE 94 3 DPL23 17 17%0/ 19 VL8 98 8 DPL21 0 0% 81 MOT 110 3 Humlv318 23 22% 38 GAR 108 3 DPL23 26 250/0 33 32.B9 98 8 DPL21 5 5% 81 PUG 108 3 Humlv318 24 23%0/ 19 T1 115 8 HUMLV801 52 50% 6 RF-TS7 96 7 DPL18 4 4% YM-1 116 8 HUMLV801 51 49% K6H6 112 8 HUMLV801 20 19% 44 K5C7 112 8 HUMLV801 20 19% 44 K5B8 112 8 HUMLV801 20 19% 44

K

5 G5 112 8 HUMLV801 20 19% 44 K4B8 112 8 HUMLV801 19 18% 44 K6F5 112 8 HUMLV801 17 16% 44 HIL 108 3 DPL23 22 21% 47 KIR 109 3 DPL23 20 19% 19 CAP 109 3 DPL23 19 18% 84 1B8 110 3 DPL23 22 21% 43 SHO 108 3 DPL23 19 18% 19 HAN 108 3 DPL23 20 19% 19 CML23 96 3 DPL23 3 3%o 12 PR-SJI 96 3 DPL23 7 7%o BAU 107 3 DPL23 9 9% TEX 99 3 DPL23 8 8%/o 19 X(PET) 107 3 DPL23 9 9% 51 DOY 106 3 DPL23 9 9% 19 COT 106 3 DPL23 13 1 2 19 Pag-1 111 3 Humlv318 5 5% 31 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96103647 Table 283: (continued) Name' aa 2 Computed Germline Diff. to 0/ diff. to Reference' family 3 gene 4 germline' germline' DIS 107 3 Humnlv318 2 19 WIT 108 3 Humlv3l8 7 19 l.RH 108 3 Humlvj18 12 11%/ 19 Si-1 108 3 Humnlv318 12 11%/ 52 DEL 108 3 H-umiv3I8 14 13 0 17 TYR 108 3 Humiv3l8 11 10%/ 19 J.RH 109 3 Humnlv3l8 13 12%/ 19 THO 112 2 DPL13 38 36%/ 26 LBV 113 1 DPI) 38 36%/ 2 WIT 112 1 D PI) 33 310/ 14 SUIT 112 2 DPL12 37 35%/ 0910 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: rearranged human heavy chain sequences Name' aa' Computed Germline Diff. to diff. to Reference' family' gene" germline' germline' 2 1/28 8E 10 MUC1-1 gF1 VHGL 1.2 HV1L1 RF-TS7 1.A15

HAILI

UC

WIL2 N89P2 mAbi 13 LS2S3-3 1523-1I2a LS2S3-5 LS2S3- 12e 1S2S3 -4 LS2S3-10 LS2S3- 12d LS2S3 -8 LS2 154

LSS

LS51 LS6 LS8 THY-29 113911 2 siPI

NEI

AND

L7 L22 L24 119 123 118 98 98 98 104 106 126 123.

123 122 123 126 125 125 125 125 125 125 125 125 125 105 125 125 125 125 122 122 122 127 127 127 124 127 VHI-13-12 VHI-13-1 2 VH I- 13-6 VH 1 -13-12 VH 1 -13-6 VH 1- 13-6 VH 1- 13-6 VH 1 -13-1 5 VH 1- 13-6 VH 1 -13-6 VH 1 13-6 VH 1 13-6 VH1I- 13-16 VH 1 -13-6 VH 1 -12-7 VH 1 -12-7 VH 1 12-7 VH 1 -12-7 VH I- 12-7 VH 1- 12-7 VH 1 -12-7 VH 1 -12-7 VH 1 -12-7 VH 1 -12-7 VH 1-12-7 VH 1 -12-7 VH 11-12-7 VHI1-12-7 VHI1-12-7 VH 1-12-7 VHI-12-1 VH-1-12-1 VHI-12-1 VHI1-12-1 VH 1- 12-1 V~I--12-i 0,00/ 0,0%/ 4.1%/ 10.20/ 2,0%/ 0,0%/ 3.10/ 1,00/ 7,10/ 5.10/ 6,10/ 10,20/ 11,20/ 10,20/ 5.,10/6 5,1 0 5,1/0 5,1%/ 5,1%/ 5,11/a 6,10/ 5,1%/ 6,1%/ 6,1%0/ 6.10/ 6,1%/ 6.10/ 7,10/ 0,00/ 10,20/ 0,00/ 0,0 0 /0 0.0%/ 0.0%/ 0,0%/ 0.0%/ 31 31 42 26 81 96 26 81 115 77 71 98 98 98 98 98 98 98 98 113 113 113 113 113 113 42 21 105 54 54 54 SUBSTITUT SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2C: (continued) Name' aa' Computed Germline Diff. to 0 diff. to Reference' family 3 gene' germline' germline' L26 133 134 136 139 141 142 VHGL 1.8 783c X17115 125 117 130 137 TNF-E7 mAblIl 111-2R

KAS

YES8c

RF-TSI

BOR'

VHGL 1.9 mAb41 0.30F305 EV1-15 mAb 112

EU

H210

TRANSGENE

CLL2- 13-3 1S7.

ALL7-1 CLL3-1 ALL56-1

AI-I

A114-1 116 119 117 118 120 120 125 101 127 127 124 120 127 120 116 122 122 121 122 123 121 101 117 127 122 117 127 104 93 97 99 87 91 85 87 94 VH1-12-1 VH1-12-i VH1-12-i VH1-12-1 VH 1-1 2-1 VH 1 12-1 VH 1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH 1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-9 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-8 VH1-12-1 VH1-12-9 VH1-12-8 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VH1-12-1 VHI-12-7 VHI-12-7 VH1 -12-7 VHI-13-8 VH1-13-6 VH1-13-8 0,0/ 0,0/o 0,00/ 0,0%/ 0,0 0 0,0 0 0,00/ 2,0%1 7.1%I 3,1%/ 7,1% 8,2%/ 8.2%/ 8,2%/ 10.2% 1 1.2%/ 1 1,2%/ 12,20/ 0,00/

O,O/

0,0/ 4 ,1/ 0,0/ l, 0 0,0/ l,0 0 O, a/a 0 0 00*0 SUBSTIUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2C: (continued) Name aa 2 Computed Germline Diff. to 0/ diff. to Reference' family' gene' germline' germlineG ALL56 15-4 CLL4-1 Au92.1 RF-TS3 Au4.1 HP1

BLI

No.13 TR 1.23

SI-I

TR1.10 Ess 1.A2 SP2 TNF-H9Gl G3DlOH TR 1.9 TR1.8 LUNmOl K1B12H 1382 ss2 No.88 TR1.6 ssl s537 s6A3 ss6 L2H7 s6BG8 s6C9 HIV-b4 HIV-bl2 L3GS 22 L2A12

PHOXIS

85 88 98 120 98 121 127 127 122 125 119 102 119 111 127 118 121 127 127 99 100 124 124 99 102 97 99 103 93 107 124 124 98 115 99 124 VH1-13-8 VH1-13-1 VH1-12-5 VH1-12-5 VH1-12-5 VH1-13-6 VH 1- 13-15 VH1-12-2 VH1-13-2 VH1-12-2 VH 1-13-12 VH1-13-15 VH1-13-6 VH 1-13-18 VH1-13-16 VH1-13-12 VH 1-12-1 VH1-13-6 VH1-12-7 VH 1-13-6 VH1-13-6 VH1 -12-1 VH1-12-1 VH1-12-7 VH1I-12-1 VH 1-12-1 VH1-12-1 VH1-13-12 VH1-13-12 VH1 -13-12 VH1-13-12 VHI-13-12 VH1-13-6 VHI-13-6 VH 13-1 5 VH 1 -12-7 5 1 0 13 5 19 23 18 14 3 15 2 19 14 24 22 23 2 2 20 19 3 0 0 0 0 0 0 21 21 1 11 3 20 5.1% 1,0 0 /a 0,00/ 1,00/a 1.0%/ 13,30 5, 1o~ 19,4% 23,5 18,40/a 14,3 3.1% 15,3% 2,0a 19,4% 14,3% 24.5/a 22,4 23,Sa/a 2,00/a 20,4a .19,4% 3,/1% 0,00/ 0.00/ 0,0 0 0,00/ 21.4%/ 21,40/ 1 ,00/o 11,20/ 3,1/a 20.4% 29 49 82 49 110 72 76 88 76 88 26 89 42 127 88 88 9 127 46 46 76 88 46 46 46 46 46 46 46 12 12 46 118 46 73 a *b a a baa.

S

a SUBSTITUTE SHEET (RULE 26) WO 97108320 PC1P6134 PCT/EP96/03647 Table 2C: (continued) Name' aa 2 Computed Germline Diff. to O/o diff. to Rfrne family' gene' germline' germline" 0* .00. 0:0.: LU Nm03 CEA4-8A HiH1O

COR

2-1151 0U

HE

CLL33 40-1 3.9 MTFC3 MTFCi 1 MTFJ1I MTFJ2 MTFUJ4 MTFUJ2 MTFC8 TD e Vq rMTF MTFUJ6

RF-KES

N 51P8 TOl 33.H 1 I SB I/D8 38P 1

BROIGM

NIE

3D6 ZM1-i 3.15 g F9 THY-32 OST577 127 129 121 127 119 124 125 120 78 88 125 125 114 114 100 100 100 125 113 114 100 107 126 119 115 101 119 119 119 126 112 110 108 120 100 122 VH 1- 1X- I VH1-12-7 VH 2-31-3 VH2-31-5 VH2-3 1-2 VH2-31-11 VH 2-31-14 VH2-31-13 VH2-31-5 VH3-1 1-5 VH3-14-4 VH 3-14-4 VH3-14-4 VH3-14-4 VH3-14-4 VH3-14-4 VH3-14-4 VH3-1 4-4 VH3-1 4-4 VH3-14-4 VH3-14-4 VH3-14-4 VH 3-14-1 VH3-13-8 VH3-13-19 VH3-]X-8 VH3-1 1-3 VH3-1 1-3 VH3-13-7 VH3- 13-26 VH3-11-3 VH3-13-26 VH3-13-8 VH 3-13-26 VH3-13-26 VH3-13-1 3 1 8,4%/ 1,0 0 9,0%/ 11,00/ 8,10/ 25.60/ 19,0 0 /a 2.00/ 7,2%0/ 21,0%/ 2 1,00/ 21,00/ 2 1,00/ 2 1.0%/ 21.00/ 2 2 00/ 23,0 0 0,0%/ 5.0%/ 100/0 9.0%/ .9,0 0 21,4%/ 10.2%/ 14,0%/ 0.0 0 13,4%/ 1 5.30/ 5,10/ 8. 2%/ 1 5,30/ 3.10/ 5.10/ 6,10/ 9 42 103 4 91 124 92 27 29 26 131 131 131 131 131 131 131 131 16 131 131 77 129 2 104 19 87 26 42 96 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' 2a 2 Computed Germline Diff. to 0/ diff. to Reference' family' gene" germline' germline'

BO

TT 125 2-A115-58

KOL

mAb6O

RF-AN

BUT

KOL-based CAMPATH- 9 1.

81 N98P1 111 17

WEA

HIL

s5A10 s6C8 s6H 12 VH 10.7 HlV-loop2 HIV-Ioop3S

TRO

SA-4B L2BS s6El 1 s6H 7 -ss 1 ss8

DOB

THY- 33

NOV

rsv13 H L3i 1 12 E8 12D0 12E 7 118 119 127 107 114 120 97 98 100 98 119 126 126 122 123 98 95 100 102 94 120 115 1 18 120 98 99 101 98 VH3-13-19 VH3-13-10 VH3-13-10 VH 3-1 3-14 VH3-13-17 VH3- 13-26 VH3-1 1-6 VH3-13-13 VHJ1-1 3-19 VH3-13-1 VH3-1 3-10 VH-3-1 3-12 VH3-13-14 VH3-13-14 VH3-13-7 VHJ1-13-7 VH3-13-7 VH3-13-14 VH3-13-7 VH 3-13-7 VH 3-13-1 VH 3- 13-1 VH3-1 3-13 VH-3- 13-13 VH3-13-13 VH3- 13-13 VH-3-13-13 VH3- 13-26 VH3-13-15 VH-3- 13-19 VH3- 13-24 VH3- 13-20 VH3- 13-19 IH 3- 13-10 VH3-13-10 1 5,3 0 11,2%/ 16,3%/ 14,3%/ 8,2%/ 13,40/ 16,30/ 13.30/ 13.30/ 12,20/ 1 5,30/ 14,3%/ 0,00/ 0,0%/ 0.00/ 0,00/0 1 6,3%/ 16,3% 16,3%/ 13,30/ 15,30/ 0.00/0 0,00/ 0,00/ 0,00/ 0, 00/ 21.4%/ 20, 14,30/ 20,4%/ 0.0%/ SUBSITIUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96I0n647 Table 2C: (continued) Name' aa' Computed Germline Diff. to 0/0 diff. to Reference' family' gene" germline' germliner L3A10 L2E 5

BUR

s4 D5 19 s5D4 s6A8 HIV- loop 13 IR 1.32 L.2B1O

TRI.S

s6H9 8 23 7 TR1.3 18/2 18/9 30P 1 HF2-1,'17 A77 B 19.7 M43 1/17 1811 7 E54 3.4 LAM BDA-VH26 E54 3.8 GL 16 4G12 A73 AL 1.3 3.A290 Ab 18 E54 3.3 3 5G G VHJ- 13-24 VH-3-13-2 VH3-13-7 V113-1 1-3 V113-13-16 VH3-13-1 VH13-13-1 V113-113-12 VH3-1 1-8 VH3-11-3 VH13-11-8 V113-13-25 VI-3 -13-1 VH-3-13-1 VI-13 -13-1 VH3-11-8 VH-3-13-10 VH-3-13-1O VH-3-13-10 VH-3-1 3-10 VH-3-13-10 VH-3 -13-10 V113-13-10 VH3-13-10 V113-13-10 VH-3 -13-10 VH 13-13-10 VH-3- 13-10 VH-3 -13-10 VH 13-13-10 VH 13-13-10 VH3- 13-10 VH 13-13-8 VH-3-13-10 V113-13-10 0,0/0 1,00/a 21,40/o 1,00/ 4,10/o 0.00/0 0,0 0 /a 17,3 0 /o 18,60/o 1.0 0 /a 2 1,60/6 0,0/ 6,1%/ 6,10/ 4.1%/ 20.6%/ 0,0%/ 0,0%/ 0,00/ 0,00/ 0,0/ 0,0%/ 0,0%0 0,0% 0,0%/ 0,0 0 1,00/ 1,0 0 2,00/a 3,1%/ 3,10/ 3, 1 0 SUBSTTLTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' aa' Computed Germline Diff. to 0/ diff. to Reference' family 2 gene" germline' germline 6 107 3 VH-3-13-1O 5 44 128 3 VH-3-13-10 5 5,1 0 16 100 N87 126 3 VH3-13-10 4 4,10% 77 ED8.4 99 3 VH3-13-10 6 2 RF-KL1 122 3 VH3-13-10 6 6j%0/ 82 AL1.1 112 3 VH3-13-10 2 117 AL3.1 1 102 3 VH3-13-10 1 1,0 0 /a 117 32.139 127 3 VH3-13-8 6 129 TK1 109 3 VH-3-13-10 2 2,0 0 /o 117 POP 123 3 VH3-13-10 8 8,2%I 115 91 21- 127 3 VH3-13-10 9 127 VD115 3 VH3-13-10 9 Vh38CI. 10 121 3 VH3-13-10 8 74 Wh380I.9 121 3 VH3-13-10 8 74 Wh380I.8 121 3 VH3-13-10 8 8,2 0 74 63P1 120 3 VH3-11-8 0 104 60P2 117 3 VH3-11-8 0 0,00/ 104 90 3 VH3-13-10 2 117 *.GF4/ 1.1 123 3 VH-3-13-10 10 10,20/o 39 Ab2l 126 3 VH-3-13-10 12 12.20/ 100 .*TD dVp 118 3 VH3-13-17 2 16 Vh8C. 11. H-31 ,%7 Vh38CI.4 119 3 VH3-13-10 8 8,20/ 74 AL3.4 104 3 VH3-13-10 1 117 FOG1I-A3 115 3 VH3-13-19 2 42 HA301 117 3 VH3-13-21 1 1,0 0 1o 81 E54 3.2 112 3 VH3-13-24 0 0,0/0 26 0.0:mAb52 128 3 VH3-13-12 2 51 0 ~S 2 H-31 mAb56 128 3 VH3-13-12 2 2,00/o 51 *mAb57 128 3 VH3-13-12 2 2.0% 51 mAbS8 128 3 VH3-13-12 2 51 rnAb59 128 3 VH-3-13-12 2 2,00/a 51 mnAbl105 128 3 VH-3-13-12 2 51 mAb 107 128 3 VH3-i3-i2 2 2,00/o 51 E53.14 110 3 VH-3-13-19 0 0.0% 0 6 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' aa' Computed Germ line 01ff.-to 0/0diff. to Reference' family' gen e 4 germline' germline 6 F13-28 106 3 VH3-13-19 1 1,00/ 94 127 3 VH-3-13-18 4 51 YSE 117 3 VH-3-13-24 6 72 3.23 106 3 VH-3-13-19 2 26 101 3 VH-3-1 3-1 3 3,1%1 N42P5 124 3 VH3-13-2 7 77 FOG11-16 110 3 VH-3-13-16 7 7 ,1%f 42 0-81 115 3 VH3-13-19 11 11.2 0 47 HIV-s8 122 3 VH-3-13-12 11 11.2% 12 mAbl114 125 3 VH-3-13-19 12 12,20/o 71 33.F 12 116 3 VH-3-13-2 4 129 4134 119 3 VH-3-1 X-3 0 101 M26 123 3 VH-3-1 X-3 0 0,00/ 103 VHGL 3.1 100 3 VH-3- IX-3 0 0,0 0 26 3.13 113 3 VH3-1X-3 1 1.0%A 26 SB5106 101 3 VH-3-1 X-6 3 3,0 0 2 RAY4 101' 3 VH-3-1 X-6 3 3,00/o 2 82-D V-D 106 3 VH-3-1IX-3 5 5,00/ 112 ***MAL 129 3 VH-3-1 X-3 5 5,00/ 72 LOC 123 3 VH-3-1X-6 5 5,00/ 72 LSF2 101 3 VH3IX-6 11 11,0%/ 2 HIB RC3 100 3 VH3-IX-6 I11 11.0% 1 56P1 119 3 VH-3-13-7 0 0,00/ 104 M72 122 3 VH-3-13-7 0 0,0 0 /0 103 M74 121 3 VH3-13-7 0 0,0 0 103 E54 3.5 105 3 VH-3-13-7 0 26 2E7 123 3 VH-3-1 3-7 0 0,0 0 63 *2P] 117 3 VH3-13-7 0 104 RF-Si2 127 3 VH-3-1 3-7 1 1.00/a 83 PR-TSi 114 3 VH-3-1 3-7 1 1,00/ KIM46H 127 3 VH3-1 3-13 0 18 3.6 108 3 VH-3-13-7 2 2,00/a 26 E53.10 107 3 VH-3-1 3-13 1 1,00/ 26 3.136 114 3 VH-3-1 3-13 1 1,00/ 108 E54 3.6 1 10 3 VH-3-1 3-13 1 1,00/a 26 FL2-2 114 3 VH-3-13-13 1 1,0 0 /a SUBSTIT1UTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2C: (continued) Name' aa' Computed Germline Diff. to 0/0 diff. to Reference' family 3 gene germline' germline' RF-S3 112 3 VH3-13-7 2 2,00/ 3.5 105 3 VH3-13-14 1 1,0/ 26 BSA3 121 3 VH-13-13 1 1.0% 73 HMST-1 119 3 VH3-13-7 3 3,1/o 130 RF-TS2 126 3 VH3-13-13 4 4,1o 82 3.12 109 3 VH3-13-15 0 0,00/ 26 19.E7 126 3 VH3-13-14 ;3 13, 1% 129 11-50 119 3 VH3-13-13 6 6,1 0 /a 130 E29.1 120 3 VH3-13-15 2 2,0/o 3.16 108 3 VH3-13-7 6 6,1 0 26 TNF-E1 117 3 VH3-13-7 7 7,1/a 42 RF-SJ 1 127 3 VH3-13-13 6 6.10 83 FOG 1-A4 116 3 VHI-3-13-7 8 8.20/6 42 TN F-A 1 117 3 VH3-13- 15 4 4.1%/a 42 PR-SJ2 107 3 VH3-13-14 8 8.200 HN.14 124 3 VH3-13-13 10 10.2% 33 CAM' 121 3 VH3-13-7 12 12,2% HIV-88 125 3 VH3-13-7 9 9,2% 12 HIV-b27 125 3 VH3-13-7 9 9.2% 12 HIV-b2 125 3 VH3-13-7 9 9.2 0 12 HIV-s4 125 3 VH3-13-7 9 9.2% 12 HIV-26 125 3 VH3-13-7 9 9,2% 12 125 3 VH3-13-7 10 10,2 12 HIV-b18 125 3 VH3-13-7 10 10,2% 12 HIV-b22 125 3 VH3-13-7 11 11,2% .12 HIV-b13 125 3 VH3-13-7 12 12,20/ 12 333 117 3 VH3-14-4 24 24,0 0 /b 24 1HI 120 3 VH3-14-4 24 24,00/ 24 1811 120 3 VH3-14-4 23 23,00/ 24 2-3 86 3 VH3-13-19 1 1,0 29 GA 110 3 VH3-13-7 19 19,40/a 36 JeB 99 3 VH3-13-14 3 3,1 0 7 GAL 110 3 VH3-13-19 10 10,2 0 /a 126 K6H6 119 3 VH3-1X-6 18 18.000 K4B8 119 3 VH3-1X-6 18 18,0% K5B8 119 3 VH3-IX-6 18 18,0% -7- 7-i SUBSTTUTE SHEET (RULE 26) WO 97/08320 PTE9,34 PCT/EP96/03647 Table 2C: (continued) Name' aa 2 Computed Germline Diff. to diff. to Reference' family 3 gene" germline' germline 6 S

S.

a.

*aaa..

a

S

a K5C7

KSGS

AL3.16 N86P2 N54P6 LAMBDA HT1 12-1 HY 18 mAb63 FS- 3 FS- S FS-7 FS-8 PR-TS2

RF-TMC

mAb216 mAb4 10.7.F91I mAbA6H4CS Ab44 6H-3C4 FS-6 FS- 2 HIGi FS-4 SA-4A

LES-C

Dl Ab2 6 TS2 265-695

WAH

268-D 58P2 mAb67 4.139 mF7 119 119 119 98 98 95 126 121 126 105 ill 107 110 105 102 122 122 124 127 124 108 114 126 105 123 119 78 126 124 115 129 122 118 128 115 Ill VH3- IX-6 VH3-IX-6 VH3-1X-6 VH-3-13-10 VH-3-13-10 VH-3-13-16 VH-4-1 1-2 VH-4-1 1-2 VH-4-1 1-2 VH-4-1 1-2 VI-14-11-2 VH-4-11-2 VH-4-1 1-2 VH4-11-2 VH-4-1 1-2 VH-4-1 1-2 VH-4-11-2 VH4-1 1-2 VI-4-1 1-2 VH-4-1 1-2 VH-4-1 1-2 VH-4-1 1-2 VH-4-11-2 VH-4-1 1-2 VH4-1 1-2 VH4-1 1-2 VI-4-1 1-9 VH-4 -3 1-4 VH-4-3 1 -12 VH-4-1 1-7 VH-4-3 1 -13 VH-4-1 1 -8 VH-4-1 1-8 VH-4-21-4 VH-4-1 1-8 VH 4-31 -13 19,00/a 1 9,0%/ 19,0%/ 3,1%/ 7,10/ 0,00/ 0.0%/ 0,00/ 0,0%/ 0,00/ 0,0% 1,00/a 1 ,0010 1,00/a 6,20/ 7 9,3% 10,3%/ 8,1%/ 16,5%/ 2 2.70/ 0,0% 1.0% 3.,1 0 117 77 77 3 43--- 86 86 86 86 is 52 100 59 86 84 62 86 125 99 58 100 110 93 6 104 108 SUBSTITUTE SHEET(RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' aa 2 Computed Germline Diff. to diff. to Reference' family'. gene" germline' germline' C 33.C9 Pag- 1 B3 1C4 C6132 N78 B2 WRD2 mAb426.4.2F20 E54 4.58 WRD6 mAb426. 12.3F 1.4 E54 4.2

WIL

COF

LAR

WAT

mAb61

WAG

RF-SJ4 E54 4.4 E55 4.A1 PR-Si 1 E54 4.23 CLL7 7-2 37P 1 AL152 30-2 EBV-21 CB- 4 CLL- 12 1-3-4 CLli 1 CORD3 CORD4 CORD8 CORD9 VH-4-21-5 VH-4-1 I- 16 VH-4-21-3 V1,4-11-8 VH-4-3 1-12 VH-4-11-9 VH-4-11-8 VH-4-11-12 VH-4-1 1-8 VH-4-11-8 VH-4-1 1-12 VH-4-11-9 VH-4-21-6 VH-4-31-13 VH-4-31-1 3 VH-4-31-1 3 VH-4-31- 13 VH-4-31--13 VH-4-3 1-4 VH4-31-12 VH4-11-7 VH4-11-7 VH-4-11-7 VH-4-1 1-7 VH-4-11-12 VH-4-11- I? VH-4-31-12 VH-5-12-i VHS1-12-1 VH 5-12 -1 VHS1-12-1 VHS-12-1 VH 5-12 -1 VH-5-12-1 VHS1-12-1 VHS1-12-1 7,1%/ 5.2%/ 8,2%/ 6.2%0/ 4,0%/ 1 1,3 0 12,4%/ 6,2%/ 2,1%/ 1,00/a 10,3%/ 4,1%/ 2,0%/ 0,0%/ 0,0%/ 2.0%/ 4.0% 5 110/ 0,0%/ 2,0%1 0,00/ 0,00/ 1,00/ 1 00 /4 0, 00% 0,0 0 4.0%/ 0.0%1 0,00/ 0,00k0 0,00/a 0,0 0 0,0 0 0,0%/ 129 53 48 77 53 52 26 52 26 26 26 26 29 104 29 13 13 13 13 17 17 17 17 17 SUBSITIUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 2C: (continued) Name' aa 2 Computed Germline Diff. to diff. to Reference' family' gene' germline' germline 6 CD+1 98 5 VH5-12-1 0 0,0/ 17 CD+3 98 5 VH5-12-1 0 0,0010 17 CD+4 98 5 VH5-12-1 0 0,0/a 17 CD-I 98 5 VH5-12-1 0 0,0/ 17 CD-S 98 5 VH5-12-1 0 17 VERG14 98 5 VH5-12-1 0 0,0 0 /a 17 PBL1 98 5 VH5-12-1 0 17 P8110 98 5 VH5-12-1 0 0,00/a 17 STRAb SA-IA 127 5 VH5-12-1 0 0,0 0 /0 125 DOB' 122 5 VH5-12-1 0 0,O/ 97 VERGS 98 5 VH5-12-1 0 0,00/a 17 PBL2 98 5 VH5-12-1 1 1,0 0 /0 17 Tul6 119 5 VH5-1-2-1 1 1,0/ 49 PBLI 2 98 5 VHS-12-i 1 1,00/a 17 CD+2 98 5 VH5-12-1 1 1,0/ 17 98 5 VHS-12-1 1 1,0/ 17 PBL9 98 5 VH5-12-1 1 1,0 0 /a 17 CORD2 98 5 VH5-12-1 2 2,00/ 17 PBL6 98 5 VH5-12-1 2 2,00/a 17 CORDS 98 5 VH5-12-1 2 2,00/a 17 CD-2 98 5 VH5-12-1 2 2,00/0 17 CORD 1 98 5 VH 5-12-1 2 2,00/a 17 CD-3 98 5 VH5-12-1 3 3,10/a 17 VERG4 98 5 VH5-12-1 3 3,1 0 %a 17 PBL13 98 .5 VH5-12-1 3 3,10% .17 PBL7 98 5 VH5-12-1 3 3,10/a 17 HAN 119 5 VH5-12-1 3 3,10 97 VERG3 98 5 VH5-12-1 3 3,10/a 17 PB13 98 5 VH5-12-1 3 3,10/a 17 VERG7 98 5 VH5-12-1 3 3.10/a 17 94 5 VH5-12-1 0 0,00/ 17 CD-4 98 5 VH5-12-1 4 4,10%a 17 98 5 VH5-12-1 4 4,10/ 17 PBL 1 98 5 VH5-12-1 4 4,10/a 17 CORD6 98 5 VH5-12-1 4 4 ,1/a 17 VERG2 98 5 VH5-12-1 5 5,10/% 17 SUBSTrTUTE SHEET (RULE 26) WO 97/09320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' aa' Computed Germline Diff. to 0/0diff. to Reference' family' gene" germline' germline" a 83 P2 VERG9 CLL6 P1318 Ab2022

CAV

HOW'

PET

ANG

KER

13 Au 2.1 WS 1 TD Vn TEL 13 5.237 VERG 1 CD4-74 257-0 CLL4 CLL8 Ab2 Vh383ex CLL3 Au59.1 TEL 16 M61 TuO P2-S1 P2-54 P 1-56 P2-53 P1-51 P 1-54 P3-69 P3-9 VH5-12-1 VHS-12-1 VH5-12-1 VH5-i2-1 VHS-12-1 VH5-12-4 VHS-i 2-4 VHS-12-4 VHS-12-4 VHS-12-4 VH5-12-4 VHS-12-4 VHS-12-1 VHS-12-4 VHS-12-1 VH5-12-4 VHS-12-1 VH5-12-1 VHS- 12-1 VHS 12-1 VHS- 12- 1 VHS- 12-1 VH5- 12-1 VHS-12-2 VHS-12-1 VH 5-12-1 VH5-12-1 VHS-12-1 VHS-i12-1 VH5-12-1 VHS-12-1 VHS-i 2-1 VHS-12-1 VHS- 12-1 VH5-12-i VHS- 12-1 0,00/ 6,1%/ 6,1% 0 7,1%/ 3,1%/ 0.0%/ 0,00/ 0,00/ 0,00/ 0,0%/ 9,2%/ 1 9,2%/ 10,2%/ 10,20/ 11,2%/ 11,2%/ 11,20/ 12,20/ 12,20/ 11,20/ 12.20/ 12,2%/ 0, 0 i 5,10/ 1 3,3%/ 11,20/ 9,2%/ 10.,2%/ 1 9,40/ 3,1%/ 4 ,1 0 4,1%/ SUBSTITUJTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 2C: (continued) Name' aal Computed Germiine Diff. to 0/0diff. to Reference' family 3 gene' germline' germline6 'S S r. 'St I. C

S

'S S S 1-185-37 1-187-29 P 1-58 P2-57 P2-55 P2-56 P2-52 P3-60 P 1-57 P1-55 MD3-4 P1-52 CLL7 L2F 10 L-3136 VH6.A12 s5A9 s6G4 ss3 6-16G1 Fi9L1G 116 M71

MLI

F19ML1 1 VH6.N I VH6.N1I1 VH6.N 12 VHG.N2 VH6.N6 VH6.N7 VH6.N8 VH6.N9 125 125 128 118 123 123 122 122 123 122 128 121 98 98 100 98 119 102 99 99 101 107 120 121 120 107 127 121 123 123 125 125 127 126 123 123 VHS-i 2-4 VH5-12-4 VH5-12-4 VHS-i 2-4 VH5-12-1 VH5-12-1 VH5-12-1 VH5-12-1 VH5-12-1 VH-5-12-i VHS-12-4 VHS-12-1 VHS5-12-1 VHS-12-1 VHS-12-1 VHS-12-1 V146-35- 1 VH6-3S-1 VH6-35- 1 VH6-35- 1 VH6-35- 1 VHG-35- 1 VH6-35- 1 VH6-35- 1 VH6-3S- 1 VH6-35- 1 VH-6-35- 1 VH6-35- 1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35- 1 V116-35- 1 VH-6-35- 1 VH6-3S- 1 VH6-35- 1 0,00/ 0,0/0 10,20/ 3,10/o 5,10/ 20.4%/ 11.20/ 8,2%/ 4,1%0/ 12,20/ 11,20/ 13,30/ 14,3%/ 1.00/ 1,00/ 1 2,90/ 1,0 0 k 0,0 0 /o 0,00/a 0,00/ 0,0%( 0,0 0 /a 0,0%/ 0,00/ 0,00/ 0.0%/ 0,0 0 0,0%/ 124 124 121 121 121 121 121 121 121 121

S

121 17 17 46 46 122 46 46 46 14 68 69 103 69 68 104 122 122 122 122 122 122 122 122 122 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCTIEP96/03647 Table 2C: (continued) Name' aa' Computed Germline 'Diff. to 0/ diff. to Reference' family 3 gene' germline' germline' *5 S S 0*

S*

S S S. 45 S. 55

S

VH6.N 10 VH6.A3 VH6.A' VH6.A4 ESS 6.16 6.17 6.6 VHGL 6.3 CB-201 VH6.N4 E54 6.4 VH6.A6 6.14 E54 6.6 6.10 E54 6.1 6.13 ESS 6.3 -ESS 6.7 E55 6.2 ESS 6.X ESS 6.11 VH6.Ai 1

AIO

6.1 FK-00i VH6.A7 HBp2 Au46.2 A431I VH6.A2 VH6.A9 VH6.A8 VH6-FF3 VH6.A1Q VYI r VH-6-35-1 VH6-35-1 VH6-35- 1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35- 1 VHG-35-1 VH6-35-i VH6-35- 1 VH6-35-1 VH6-35-i VH 6-35-1 VH6-3S- 1 VH6-3S-1 VH-6-35-1 VH-6-35-1 VH-6-35- 1 VH6-35- 1 VH-6-35-1 VH6-35- 1 VH6-35-1 VH 6-35-1 VH 6-35-1 VHG-35- 1 VH6-35-1 VH6-35- 1 VH6-35-1 VH6-35-1 VH-6-35-1 VH6-35- 1 VH-6-35-1 VH6-35-1 VH 6-35-1 VH6-35-1 VH6-35-1

A*

0,00/0 0,00/0 0,0%/ 0,000 0,00/ 0,0 0 /c 0,0 0 /a 0,0%/ 0.0 0 /0 0,0 0 0 1,00/0 1,00/0 1,0 0 /o 1 .00/c 1,00/c 2,0 0 /b 2.00/ 3,00/o 4,0 0 /b 5,00/0 5,0/0 5,00/ 7,90/ 9,9 0 /0 11,9 0 /0 555555

S

5S55 .5 55.55.

S

S

SUBSTITUT SHEET (RULE 26) WO 97/09320 PCr/EP96103647 Table 2C: (continued) Name' aa Computed family 3 Germline gene Diff. to %/diff. to germline' germline 6 Reference' VH6-EB1O VH6-E6 VH6-FE2 VHG-EE6 VH6-FD1O VH6-EX8 VH6-FG9 1 VH6-EC8 1 VH6-E10 VH6-FF1 1 VH6-FD2 1 CLO 17-2 VH6-B11 VH6-B41 JU17 VH6-BD9 VH6-BB9 9 117 119 121 116 118 113 121 16 22 20 22 15 38 34 33 02 16 14 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35-1 VH6-35- 1 VHG-35- 1 VH6-35-1 VH6-35-1 VH6-35- 1 VH6-35-1 VH6-35- 1 VH6-35- 1 VH6-35- 1 VH6-35-1 VH6-35-1 3 3,0 0 /o 6 5,9% 6 5,9/ 6 5,9/o 6 5,9% 6 5,9/ 8 7 ,9/ 9 8,9% 9 8 ,9/ 10 9,9 11 10,9% 11 10,9% 4 40 0 /a 4 4,0% 7 6,9% 3 3,O 0 11 10,9% 12 11,9% 123 123 123 123 123 123 123 123 123 123 123 123 29 123 123 114 123 123 VH6-35- 1 12 l1.9 0 /a 123 SUBSITUME SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 3A: assignment of rearranged V kappa sequences to their germline counterparts Family' Name Rearranged' Sum I Vkl-l 28 1 Vk 1-2 0 1 Vk 1-3 1Vk 1-4 0 7 IVkl-6 0 1Vkl-7 0 1Vkl-8 2 1 Vkl-9 9 1 Vkl-10 0 IVkl-llI I Vk1- 12 7 1 Vkl 13 1 Vk1- 14 7 1 Vk1- 15 2 1 Vkl-l6 2 1 Vkl-17 16 I Vkl-18I I Vk1- 19 33 j Vk 1-201 1 Vkl1-211 1 Vkl1-22 0 1 Vk 1-23 0 119 entries *9* Vk.2-1 Vk2-2 Vk2-3 Vk2-4 Vk2-6 Vk2-7 Vk2-8 Vk2-9 Vk2-l0 Vk2-I I Vk2-12 Vk3-l Vk3-2 0 0 0 0 06 0 0 0 7 0 25 entries SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 3A: (continued) PCT/EP96/03647 Family' 3 3 3 .3 3 3 4 6 6 7 Name Vk3-3 Vk3-4 Vk3-5 Vk3-6 Vk3.-7 Vk3- Vlc4- I Vk5-1 Vk6-1 Vk6-2 Vk7-1 Rearranged' 115 0 0

I

40 33 1 0 0 0 Sum .192 entries 33 entries I entry o entries o eniries 4 4 4 444444 4 4 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 3B: assignment of rearranged V lambda sequences to their germline counterparts Family' Name Rearranged 2 Sum 1 DPL1 I 1 0P1.2 14 1 DPL3 6 1 DPL-4 1 1 HUMILV1 17 4 1 DPLS 13 1 DPI-6 0 1 DPL7 0 1DPL-8 3 1 DPL-9 0 42 entries 2 DPL10 2 VLA M BDA 2.1 0 2 DPL1 1 23 2 DPL12 2 DPL13 0 2 DPL14 0 43 entries 3 DPL16 3 DPL23 19 3 Humlv3l8 9 38 entries 7 DPL18 1 7 DPL19 0 1 entries 8 .DPI.2 2 8 HUMLV801 6 8 entries 9 DPL22 0 0 en tries unassigned 0PL24 0 0 entries gVLX-4.4 0 0 en tries

G

a.

a.

a a a. a.

a a a a a SUBSTITUTE SHEET (RULE 26) WO 97/08320 .PCT/EP96/0364 7 Table 3C: assignment Of rearranged V heavy chain sequences to their germline counterparts Family' Name Rearranged' Sum 1 VH 1-12-1 38 1 VHI-12-8 2 1 VH 1-12-2 2 1 VH 1-12-9 2 1 VHI-12-3 0 1 VH 1-12-4 0 1 VHI-12-5 3 1 VH I- 12-6 0 1 VHI-12-7 23 1VH-1-13-1

I

1VHI-13-2 1 1 VH I- 13-3 0 1 VHI-13-4 0 1 VHI-13-5 0 1 VI-13-6 17 1 VH I -A3-7 0 1 VH-1-13-8 3 1 VH1I- 13-9 0 1 VHI-13-10 0 *V I 1 V113-11 0 1 VHI-13-12 VHI-13-.17 0 1 VHI-13-18 0 1 VHI-13-19 4 I VH1-13-16 2 0enre 1 V-1-21-17 0 2 VI-131-1 1 1 VI-131-1 0 1 V-1-3X-1 13nre 2 VH-2-31-4 0 2 VH-2-31-5 0 *a2 VH12-31-6 0 2 VH-2-31-7 0 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Table 3C: (continued) Family' Name Rearranged' Sum 2 VH-2-31-14 1 2 VH2-31-8 0 2 VH2-31-9 0 2 VH-2-31-10 0 2 VH-2-31-11 1 2 VH2-31-12 0 2 VH-2-31-13 1 7 entries 3 VH-3-1 1-1 0 3 VH3-11-2 0 3 VH3-11-3 3 VH-3-11-4 0 3 VH-3-11-5 1 3 VH-3-11-6 1 3. VH3-11-7 0 3 VH3-11-8 3 VH-3-13-11 9 3 VH-3-13-2 3 3 VH3-13-3 0 3 HS.- 3 VH3-13-4 0 0 00 .1.3 VH3-13-5 0 *3 VH3-13-6 0 0 3 VH3-13-9 32 3 VH3-13-80 46 3 VH3-13-11 0 3 VH3-13-10 46 S3 VH3-13-13 07 3 VH3-13-12 81 3 VH3-13-13 17 3 VH3-13-16 8 3 VH-3-13-17 4 3 VH3-13-16 3 3 VH3-1317 23 3 VH3-13-20 1 3 VH3-13-1 13 3 VH3-13-20 1 SUBSITUTE SHEET (RULE 26) WO 97108320 Table 3C: (continued) PCT/EP96/03647 Family' Name Rearranged' Sum 3 VH3-13-23 0 3 VH3-13-24 4 3 VH3-13-25 1 3 VH3-13-26 6 3 VH3-14-1 1 3 VH3-14-4 3 VH3-14-2 0 3 VH3-14-3 0 3 VH3-1X-1 0 3 VH3-1X-2 0 3 VH3-1X-3 6 3 VH3-1X-4 0 3 VH3-1X-5 0 3 VH3-1X-6 11 3 VH3-1X-7 0 3 VH3-1X-8 1 3 VH3-1X-9 0 212 entries 4 VH4-1 1-1 0 4 VH4-11-2 4 VH4-11-3 0 4 VH-4-11-4 0 4 VH4-11-5 0 4 VH-4-1 I-6 0 4 VH4-11-7 4 VH4-11-8 7 4 VH4-11-9 3 4 VH4-1 1-10 0 4 VH4-1 1-11 0 4 VH4-11-12 4 4 VH4-11-13 0 4 VH-4-11-14 0 4 VH-4-1 1-15 0 4 VH-4-11-16 1 4 VH4-21-1 0 4 VH4-21-2 0 4 VH4-21-3 1 4 VH-4-21-4 1

S.

0O S 0O@)

SS

@6 6 0O

S.

0 S 0S SO S. S 6

S

S

S

S

S

050050 6 0 SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96103647 Table 3C: (continued) Family' Name Rearranged' Sum 4 VH4-21-5 1 4 VH4-21-6 1 4 VH-4-21-7 0 4 VH4-21-8 0 4 VH-4-21-9 0 4 VH4-31 -1 0 4 VH-4-31-2 0 4 VH-4-31-3 0 4 VH4-31-4 2 4 VH4-31-5 0 4 VH4-31-6 0 4 VH-4-31-7 0 4 VH4-31-8 0 4 VH-4-31-9 0 4 VH4-31 -10 0 4 VH4-31-11 0 4 VH4-31-12 4 .4 VH4-31-13 7 *4 VH4-31-14 0 VH4-31 -15 0 *4 VH4-3116 0 4 VH4-31-17 0 4 VH4-31-18 0 4 VH4-31-19 0 *4 VH4-31-20 0 57 entries VH5-12-1 82 VH5-12-2 I VHS-12-3 0 VH5-12-4 14 97 entries 6 VH-6-35-1 74 74 entries 0:* SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCTIEP96IO3647 Table 4A: Analysis of V kappa subgroup 1 I F-ramework I amino acid' c L11 10 IcnC I A ii.H102:1 B11 C1 D 641 F 1 6 1

H

K6 51 SL.- 6: 21: 96! 1 66: N. P 103 1 21 Q 62: 8811 £89: 102 8 13; 103.

T *i88:1 V98: 2: 98: un no n LO OOYO f- 0 unkownpid 4Q) ~~21 1 y..

SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4A: Analysis of V kappa subgroup1 PCT/EP96/03647 amino acid' 'r :4 7 U)N (0n 1.OCW r' B1 C ~105: 1 K 2:1 F 2010

H

K 0 0 0 0 eloMca0 0 .:0 N~a~ pie 4 4 0 SUSPUESEE RL 6 WO 97108320 Table 4A: Analysis of V kappa subgroup 1 PCT/EP96/03647

CDRI

amino acid' Lu ILTN r: M =nM M C n e I A 1 11 421 B1 1 C1 D 25: 11 57:1 E 121 F 1 6: G 251 73: 4 2 9 8: 1 4;1 K T 9 L 2 1 101:

M

N 6! 16421 P 102: o98: 103: 2: R 16: 3 2 31 S41 2::57323 1 1 7 4: 4: V 1 4 11 W 2 1 104: x1 Y 1 60: 98' 105' 105i unknown()3 not sequenced 1 1 1 1 0 .3 *.0 sum of seqI 1.io1051 1050 10S~ 105~ 104 104: 1041 104~ 1041 1041 1041 1041 1041 1fl4~ oomcaa mcaa' rel. oomcaa' pos occupied': 105: 105: 41: 98 571 421 60: 101: 50i 104i 981. 98:: 103: 95' 102: S i~ I ~SN L~ L N Q~y K P 6 12: 111 9 4 8 1: 25: 2 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 4A: Analysis of V kappa subgroup 1 Framework 11IF CDRI11 amino acid' n wc a'T L O n n A 94 50

B

C

D 21 1 1 1 F 1: 3 1 H 2: K 95 861 2; M 2 1 111 6 K 95861 516; 1994 2 V 8 9:10: 1 x 1041 note5uece 11 1 99 1: 1 a a sum Of sea' 104:: 1041 104:: 104:1 104: 104: 1031 102 102: 103: 104: 104: 104: 1041 104: oomcaa mcaa' rel. oomcaa I 1001 95: 94; 104: 86: 891 103: 100: 92: 50: 95: 99: 41: 101: 62: G K A P K' Li L I Y A A S S L 0Q .e pos occupied'1 2: 6: 31 81 61 11 2: 410::6: 6: 9 3 6 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4A: Analysis of V kappa subgroup1 PCT/EP96/03647 amino acid' L' SOG __LS L W W O (O A 3: 2 1 1 1 B1 D 67: E 130: F 11031. G21105i 105S 4: 101: 1021 H 3: 1 31 411 3 L1 M1 N 6: P 1101: 2 R 1,103.- 1 2: 68::tS268 103: 98: 961 100: T 19:1 1 2; 3: 101 v 99 9 x 1112: unknown(? not sequenced sum of seql 105 105: 105: 1051 105i 105i 105: 1051 105! 1051 1051 1051 1051 105: 105: *9oomcaa' 687 105 99: 101: 103: 1031 1031 981 1051 961 101: 100: 1021 1011 67: *mcaa 4 S G V P S R F S 6 G S G TOD posoccupied': 10: 1: 4 4: 2: 31 3; 5: i 4 4 4 4 7 SU BSTITUTE S HEET (RU LE 26) WO 97/08320 Table 4A: Analysis of V kappa subgroup1 PCT/EP96/03647 Framework IIl amino acid' co cn c) -n .Z A231! 101, B 13: 2: D 116: 101: E 83: F 21102 1 2 73: G 4:1 2

H

99: 5 171

K

L 81103: 11

M

N 7: 4 P 97: R 21 2: S 2:1 86: 94: 4:1 T 98 102: 21 1 97V V1 2: 4: 1* U 1 Y 1: unknown (P) [notsequen e 1 1 1 1 1 1 1 1 2: 2: 2: 2 2: 2: 3 boo* .094 .e to.s9 &woe a .00.

0 *0 0 sum of seq' oomcaa 3 inca a 104:. 104: 104: 104:: 104i 104: 104: 104: 103! 1031 103: 103: 1031 103; 1021 1021 98: 81 102991 86: 94 103: 971 97: 83: 101 731 101: 97T F T L T I S S L Q P E D F A T rel. oomcaa&6 -e i pos occupied; 3 4: 3: 3: 3: 7: 5 21 4: S; 21 5: 2: 6 SUBSITIUTIE SHEET (RULE 26) WO 97/08320 Table 4A: Analysis of V kappa subgroup 1 PCT/EP96/03647 CDR IlI amino acid' C' C )a A 1 7: 1 5: 1- B 3! C 1021 D 2 3; 5: 1 F 7 3: 13: G 11 2: 1 1 H 1 4 6 7':3 11 4: 1 2: 1 K 17:1 L 7! 61 2 1 18:

M

N 61 3 1 19: 1 P 1 82! 6: 090:: 86:: 1. 2 R 12: 2: 2 7: 3 58: 510: T 3 11 15: 2 x Y 1011 93: 42: 32: 1: 23i 8 2 8 8 8 9 89: 89: 89: nosq enced 2 3: 3 2: 2: 1 1 1 11 4 16' 16; 16: 16: 16:1 sum of seq 1031 1021 1021 1031 1031 104 104: 104: 1041 1011 891891 891 8 9: 8 9 8 9 oomcaa 3 101! 93: 1021 90. 86. 42: 32: 58: 25: 828289 98 89; .ca y 82 C8O 89 89 89T S4. SUBSiTUT SHE (RL 26)YY S WO 97/08320 Table 4A: Analysis of V kappa subgroup1 PCT/EP96/03647 Framework IV amino acid' cn 2 -h UM A 1627 B 1119 C 209 D 1 459 E 2: 65: 258 F 6: 86: 2: 451 G 871 29: 8 7! 2 894 H 21 1 5: 72! 606 K 11 7 791 480 L 18 1 2 2 4: 2 793 M 1 5: 77 N 121 232 P 6: 7: 1620 Q 148.1 865 R 6: 6 2 70 413 S 221 1636 T 2:8 2 1873 2: 1021 V 2: 1 63: 3 440 W 15: 141 X 14 Y 16:1 564 4- 41 851 1 1250 unknown ()7 not sequenced 161 16 18: 18: 18: 181 181 18: 19: 19- 20 20: 201 31 589 sum of seq' 891891 871 87: 871 871 87: 87: 861 86: 85: 85:851 74: oomcaa, 188 2868 7:48!871871771 6 3: 6S 7 2: 8 5: 7 9 701 mcaa, L TFGG GT IK V EI K R rel. oomcaa' .i ec pos occupied": 17' 7 2: 11 5: 1 1: 4: 3: 5: 6: 1: 4 4: 9 99 99*9** 9 *.99 9*99*9 9 SUBSTITE SHEET (RULE 26) WO 97/08320 Table 48: Analysis of V kappa subgroup 2 PCT/EP96/03647 F- Framework I amino acid' cN-~ -0 L D m -n 2 i !L2 C

A

C

D

E

F 22: 14: 3::1 0*

C

G

H

8: 22:

K

L 3: 117 1 6: M 1

N

18; 15: 2 2: Q18: 7

R

S 18 17 12 2: T 17: 21: V 6!17! 11 18:

Y

unknown()1 not sequenced 5: 5: 5: 5$ 4: 4: 4 4: C 4 4: 4: 4: 1 1 sumnof seql 17: 17:; 17: 17T 18: 18: 18: 18: 18; 18: 18: 18: 18: 2 1 21: 22: 22: 22:22:22:22: oomcaal 14: 8: 17: 15: 17: 18: 18: 18: 17: 17: 18: 18: 18:21: 15:22: 15 22:22: 22:22: mcaa' D IV M TI Q S IP I SI L IP V T P G E P AS I rel. oomcaa' a' :C i0 )i0C 0 o. 0 000 CN 0)C 0 OCOj-5):CO 0: 0D 0D O occupied' 2: 3 1 3 1; 1 1 1 2 2 1 0 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4B: Analysis of V kappa subgroup 2 PCT/EP96/03647

CDRI

amino acid' C' (0N C'N C' '.LI <C w u- C. c-0c) m'

A

C

E

F

Q 7

G

H

K

L

M

1 2: 16 1 1 22: 13: 22:

S

N 10: 7:12: 9

P

R 2 1: 2: S 21 2 222 2 2: 12: 19 1 I T 8: w 22.

no Weune

S

SuIM of seq' oomcaa' incaa' rel. oomcaa& POS Occupied"' 22 22 2::2 2::2 2::2 2::2 2;:2 2 22 2 2: 2 2: 2 2: 2 2: 2 2: 2 2 i2 2 2 2: 22: 2 2: 2 2 2 2: 2 2 2 1: 2 2:21-: 2 2:2 2: 21 22. 2 2. 13: 16: 19: 22: 10 22: 11: 12; 21: 2 2 11: 2 2 S CR SS IQS IL LIH IS N G Y NY LD W:Y 0 Oo SO~ i:,a C; 0! a o Co aCoi0:a m Lb: 5: 5 io if 0) Lfl o: 0 2 i 2: 1~ 1 3: 4 3: 1: 5 1 5: 4: 2 1 4 2' SUBSTITUTE SHEET (RULE 26) (9z flflv) .L HS un i i i asfS I L A I t, t L c iL L :z t :Z ,p.idnDxosod LO O:0 :0 .o 's n j P, o c) 0 w o ,ee:)woo -i C) 0 0 0 .S V ~N S 0 1 A I 1 :zz Iz :ti :81 iiz iz tI Ii EU LZ :t7L ii d S0 9 d i Ii z z z z z S. Iz 9 t eez) ,ee,)uwoo £b~s jo wuns zIi zi Li z i z i z i ii Zii'i~z L i za Lx :9 Aiii :zzi

ZN

In -:9 Lf U, U II zQ iiz djO~e Z dno16qns eddelj A JO sisAleuV :qt, )Iqei OrESOIL6 OM LI'90Of96da13d WO 97/08320 Table 48: Analysis of V kappa subgroup 2 PCT/EP96/03647 Framework III amino acid' i 2no LT. 33 7. c.N m. C-0CS c c~o 0. w r, r-.

A

B

C

D

E

F

G

H

K

L

1 9 2 1 9 I

M

N

P, 2 2: R 20: H 2 211 20:1 T 1 22: 211 V 22: 1 21 x

Y

unknown()1 not sequenced111111 sum of seq' 22i 2 2: 2 2' 22! 2 2 2 2: 22:. 2 2: 22: 22.: 2 2. 22:: 2 2 2 2 2 2 2 1 2 1: 21. 2 1. 21: 2 1 oorncaa *22 22:: 22: 2 01 21 22: 21: 21: 22; 22 21: 22: 22: 22121 i2 1119,21!20:20:21: mca V P D R F S G SG S G T D F T L K I S R V 0 00000 0000 0 0 0: 0D rel. oomcaa' 6CC C 00 Co -C0 6Co 00: O0 0a:0: pos occupiedr: 1: 1: 1: 3: 2 1: 2: 2 1 2: 1: 1 1 1 1 1 1 2; 2: 1: 9 999999 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4B3: Analysis of V kappa subgroup 2 PCTIEP96/03647 I CDR III amino acld r" CO Co oC c O OCo o M nc m A 2 0: 14:: 1: B11 D~ 1121 E 19 2 1:

F

G121: 6:1 2: H 1 7:

K

L 12: 21 M 21:

N

P 1 2161! 2 13: R1 T 8: 7 V 21: 19: 6 7 not sequenced 1~ 1 1 1 1 1: 1 1 1 2H H5H H51: 2: 555 t sum of seq' 21:: 21 21 21 21;21; 2 1:21: 2121:2 21. 2 1:21: 21;: 21120: 17: 17: 17: 17: oomcaa :19 20: 20: 21:2121 19: 2 1 1:21: 0 14: 12; 13: 7: 16: 14; 17: 17; 17: I mcaa' *E A E D VG V YY C:M Q:A L Q: TP i 00: e,0 0;0 U, 0 C :0 :0 c0 0) 00 0: re~oma C- C) 0000 0 0 pos occupied"' 3: 2: 2: 1 1 3: I 1 1 1 2 3 3 3: 7 3 3 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 48: Analysis of V kappa subgroup 2 PCT/EP96/03647 Framework IV amino acid' LU jo Or-) cn p Sum A 71 B 13 C 43 D 112 E13: 71 F 1 17: 72 G17: 2:16 1 233 H 26 1 3: 14: 94 K 12: 13:: 66 M 37 N 56 -P 1 Q 114: 159 R 4: 12 126 S 325 T 17: 16: 140 V 5: 146 31 x 3 Y 7: 123 17:17 17 1313 unknown Q?)2 4 notsequenced 5: 5: 51 5: 5: 66 66: 6: 7: 8 9 910 211 sum of seq' 1 17: 17: 17: 17: 17: 17: 16: 16: 16: 16: 16: 15: 14: 13: 13: 12: oomcaa' 171 7 17: 17: 17: 14: 161 16: 12: 11 13: 14 13 13 12 mcaa, Y T iF 1GQG T KL E K R 0 .e 00 rel. oomcaal CD 00 8 0 0 00 00 0 LMfla) r: >:C pos occupied" 1: 1: 7 1 1 2: 1 1 2: 21 3: 1: 1! 1 9

CS**

105 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96/03647 Framework I amino acid' Lo~ "0 r- 0

L~~

A

C

E

H

K

L

.M

N

P

Q

R

S

T

V 5: 2 27: 2: 11 1 4 757: 82.

A

A..

a a .2 w j 117: 14 1.22.1 x

Y

II

S b 4. unknown 4 n ot sequencd__ sum of seq' oomcaa' mcaa' rel. oomcaal pos occupied' 88; 88::117::118i 118: 123: 123:124: 126; 149:151: 152:152: 152: 152; 152: 76: 75! 891104: 117: 1231 119:124: 82;:147:150 101914 4 ElI V L T Q S P G T L S L S PG 6: 6; 3: 3: 4: 1: 4: 3: 2 2 3 4 1 SUB3STITUT S HEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 PCTIEP96/03647

CDRI

A

B

C

D

E

F

G

H

K

L

M

N

P

a

R

S

T

v

Y

178! 2: 6 1 18V7 14159 175~~ 71 11 S4 4 4 4 1 72 189-.: 1 4. A? A') Sunknown(? not sequenced4. 4 .4 sum of seqI Ioca omcaa rel. oomcaal occupied,: 15311811 1821 182:1821 182::181: 182: 182:181: 181:121218 8 8 146: 175 178:174: 173:180:181: 176 1615198 12121212 E R A TjL S C R A SQ .3 7: 2. 4 3 3 3: 5: 6: 6 8 1 1 SUBSTITUTE SHEET (RULE 26) WO 97/083209 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96/03647 amino acid' U. m A LOJ w~ r' co c A 1 1181: 4. E11 F 17 1 G 21 7: 3 1 2: 1184: H 12:1 12: 1 1 2 4: 4 1 1 K 1:1153: L 8 1 176:- 3: 2: N 3: 12: 25: 32: P ~170: 1183:167: 1. 181: R10 318i 1 1 27::5: S 72; 86::151::118: 4: T 1 3 8 1v .7 6:681 1 7i 3 2: W 185:: x Y 1: 1:115: !183: 182: unknown()1 ntquenced sum of seq' ::182::182::182::181:181::182: 183:184: 185: 185: 185: 185: 184; 184:184: 184; oomcaa' 1182: 76: 86::151: 118! 115S 176: 181: 185:1831183::167.- 153 170: 184: 181: mcaa' v S S S Y L A W Y Q OQK P G 0 rel. oomcaal i C-2o 2? C CN M n C1 to: O C) Cn;CN 0D CO pos occupied': 1 6: 11~ 10: 13; 12: 2: 3 1 3 2 4 6: 6: 1 3: .4 4 4 4..

SUBSTITUTE SHEET (RULE 26) WO 97108320 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96103647 *rk 11 CDR 11 LO. e f W 00 a) C14. t q- Ln CO-.

amino acid' V 4- r t -z r L Ln Ln Lo L U LO Ln Ln A 176:; 4: 4T 17 6: 1.

D 4 3 2: 4:

E

F 1 41. G 125: 2: 10::179: H 9:1 178:1 168: K 7 71 L 1 179::174: 1. M 311 N 1153 2: P 5:184 2! 221 Q R 182i 1! 4180: S3: 6: 4:1797 4; 1 T 31 11 24 4: 164 2: V 3 :3 9 3 19: 31 151 G. .4 x Y 165: 2 unknown(? P) 1 not sequenced a a a.e a sum of seq~ oom caal 18 8 185: 1 8j: I t3j: 183: 183:183: 183--:183:183:183: 185: 185: 185: 185: 1761:184 1821: 179:: 174::1 78:1 1651125: 1471 179: 74;1180: 176: 164'179: 168: mcaa, A P R L LI G S A rel. oomcaa' C O~ M: CO O Ln i 0 O oi 0 0o C L a pos occupied': 3: 21 31: 3 2: 4 7 66 5: 7: 3 3:: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96/03647 Framework III amino acid' TO CD CD MDZ0 W)'o LO .0c r' Z A 68: 3: 5 3: 1 3:

B

D 11121 11152: E 30::3 F 183: 183: 2: G 184: 13178: 177

H

K..

7~ R 182 2:1 1 17 21 7 7 7 n n w n 7 e .r 4* a a a a a aUIII vI acq It5b: 185: 185: 185: 184: 184 184 184 184 oomcaa :177!11 1821 183: 180: 184: 179: 178: 185: 177: 177 :152:183:172: 182: 179; mcaa' P D R F S G S G S G T D F T L T CO a) C:C 0) a a) 0 pos occupied" 3: 5: 3i 3: 3 21 41 5:1 i 5: 4: 2: 5 2: 3: 110 SUBSTITUTE- SHEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96/03647 amino acid' rn rwPZ'o r c, o '0 '0 -O "'CO CO C0 C'

A

B

C

D

E

F

G

H

3: 17 4- 1 78 221 143 17 21 18178 1 9 4 49 9 9* 9 9 9 9 9 9***99 K1 L 17 8;1 7;1 M N 1 P 149: Q 34: 11181:1551 R 11131 S 11691 651 3 41 2: T 8411 8: V 4: 6: 131591 7 x 1 Y 1 183:176:- 1 2: d 4. 4 unknown() not sequenced 999999 9 (9 9 '909999 4 0 .9999*9 sum of seq 184::84::184::1841184 184::182::184: 184;, 18411841 184: 184::183::1831 183: oomcaal 17811691 111: 178: 149 149: 175::182: 178::1741159::183:176:182:181:155: mcaa' I S R L E P E ID F A V Y Y C 0 Q rel. oomcaa l ~4 a) LOl (0 (n L:O n) a) Cn: to :C o C n n pos occupied'i 4: 5 5: 2: 3: 4~ 3 6: 7. 5: 2: 3;.8: SUBSTTTE SHEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 PCT/EP96/03647 CDR IIIl amnino acid' m cn a)m M C A 1 8: 3: 3

B

C 2:1 2: 0 8;:5:1 E 2 1 F 5 2 7: 166: G 1104::15: 1 2: 1 16641: H 4::1 2: 1 4: K 2 1 L 2: 7: 5: 42: *M 11 2 N 28: 7 1:1 P 1:13 924: 72: 9: Q 113 1 3 :114: R 3 4: 2: 3. 2 2: 19: S 21 331 5 810 2: 15: 2 1 8: T 2:;1311 2 1 154: V 31 2: W 69: 2 4: x Y 134: 1 1 43: 3 3i. 7: 2..?1271 167; 69:19: 169: 169: 8: 1 1 1 unknown (P) [not sequenced 14: 14: 14: 14* 14:' 14;: 1 1 7: 16' 16 16: t

S

S.

S

(555.

S

(S S

S

4 S.

S

S

sumr of eqornca a' mcaa' rel. oomcaal pos occupied"~ Ib169:19 169J ib 169 1b69 169: 1665. 167* 167 167: 134 104; 71; 102 13 127::167; 169: 169::169: 169: 43; 154; 166; 166; 114* Y G N S P Y T F GO0 I~ a' 0 C0 C5 0 Bae a.i a,62 0M 0) C) 0: 0: 0n en 0n (M CO mr LO0 3~ 1~ 1 8: 11: 12: 2: I I !1 I: 18: 5: 2: 2: 6: I I 112.

SUBSTITUT SHEET (RULE 26) WO 97/08320 Table 4C: Analysis of V kappa subgroup 3 Framework IV PCT/EP96/03647 r- C-n V LO) W(0 amino acid' 0 0 0D 0 0 0 0 C Sum A 14 B2 C37 D 2 3:;6 141 7659 G 166110 H 16 K 152: 157: 489 L 4 2 21596 M 3:3 N 3i .25 .9 1314 R 9! 2! 4:134 1326 w 287 159 y 11 164611 6 2 Wteune 61 51 61 71.4 3 ~umoseq 67 67 18 17 16 167166166:138 16 15 141 143. 16 157.13 mcx G T K V E I I 9 9* 9 9 9 499999 9

L

rel.oomcaa" 02 CD 0 0 (0 0 pos occupied'*: 2: 5S 7 I S 7 1 4 SUB STITUTE SHEET (RULE 26) WO 97/08320 -PCT/EP96/03647 Table 4D): Analysis of V kappa sub group 4 1 Framework I amino acid' r-4 en~ t~ LI) w. N L2) T. A 24:

B

C11 D 2 5: 26: E 2

F.

G 124:

H

26: K1 L 126: 26: M ~24: N1 P 26: Q 1 R 2 6: S26 1 2 5: 26 1 T 26: V 25 1 26' x unknown(? notsequenced 7: 7: 7: 7 7 7: 7 7: 7 7 7; 7 7: 7: 7 7: 7: 7 9 9 4 *9*9 9 p

C

*0pp** sum of seq? oomcaa) 26 26: 2 6: 26I 26 2 26 26: 2 6 2 6: 2 6:2 26 26 26 26 2 6 2F G 2 5 1 2 61 2 24: 26:: 2 26: 26: 2 6: 25: 26' 24: 2 6: 2 6; 2 6: 2 4: 2 51 2 6:: o t mcaa' D I IV M T a S P D L V rel. oomcaa' o* I 000 0 0 D: W 0. C) 00 0 pos occupied" 11 3: 1~ 2: i V 2: 1 3: SUBSITUTIE SHEET (RULE 26) LGE R 0 CN 4 0 13::21 WO 97/08320 Table 4D): Analysis of V kappa subgroup 4 PCT/EP96/03647

CDRI

OC0 N M Ln CD CO MoC C%(4 (N N (NJ C4 N (4 C%4 M amino acid' B C 331

E

G

H

33 2 1 2 31; N 26 Y T 2631 V. 28....2 w 32 321 unknown. n Tseune 7 76 7 S

S

S.

S S

*S

5

S

S

S

S

SOS...

S

555C*

S

sum or seq' oomcaa') mcaa' rel. oomcaa' pos occupied" 26: 265 265 26 33: 33: 33: 33: 33: 33: 33: 33; 33: 33: 33: 33: 33: 33 2 6: 26: 26: 2 6: 331 331 31i 3 3 32:3 3: 2 8:31 3 2: 32! 3 2 30: 31: A T II N C K S Q SV LY SS N N K CD: C: 0 00 0 80 al: 00:C)0: 0i 0 00: 1 i3 1 1 5: 2: 2 3: 1 4: SIUBSTiTUT SHEET (RULE 26) WO 97/08320 Table 4D): Analysis of V kappa subgroup 4 PCT/EP96/03647 Framework 11 amino acid' e4 en tfl CD r- CO M~ 0) C14M tL W f A 3 2: 2:

B

E1

F

G 32; H 2 0e .5 0 S 9S** S@ *4 4 S

S

6 K 33: 32: L 33: 29:33: N 333 31; 31: 2 3333333 3333.. 33 33. 3333333333333333....

ooSa 33 33 33 233 133 13 22 33 N L A W K p K2L 0T 0 posocupid 1 1 1 2 2 1 2 2 2 1 2 1 gee...

S

SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 40): Analysis of V kappa subgroup 4 PCT/EP96/03647 I ~CDR 11 amino acid' M~ 0 Cj M~ Lfl W a) 0 rj M~ V- LO L Lnl U Lfn U) U) U) U) U) L) It W (0 W. W. W. W. Wf A

B

D 33: E 32: F 3 3 3 13 3 33

H

K

L

M

N 2: P 133: 0@ 09 9 6000 *0 9 0 9*

S.

S S 0S S* @9 S 9 9 000099

S

9 099909 9 9 9 @06099

S

9900 9 0055

S.

090000 0

S

000000 6 R331 32: S 1 31 11 33 32: 33 T :212 9! 1 33; x Y 33Z unknown(7 not sequenced 00 mca a' mcaa' rel. oomcaa' pos occupied', 33 j s~ Jj JJi 33i 31~ 33: 331 3]1 331 33 33; 33: 33: 3 33 3:3 0:3 11 2 93 3:3233 33 33 33 33323332:33:33:33: YW:A STIR E S GV P DR FSG .e 00 i o'4 2 o 0; <5 0 C0 0 0 0S S 0o al 0: 0 0: 0 r. 0 0 0 r 00 0 1 1 32 3 4: 1 2 1 1 2: 1 2 1 L SUBSTIIITUTI SHEET(RULE 26) WO 97108320 Table 4D): Analysis of V kappa subgroup 4 PCT/EP96103647 Framework III amino acid' rw CO 01 C)(.0Ir rl- r CO~ CO CO

A

C

E

F

G

H

K

L

M

N

P

Q

R

S

T

x Y 3 32: 32 333 333 3221 1* 33: 33:3 1 301,3392: 33. 1 331 4 unknown Q?) not sequenced sum of seqI 3 3 33 3 3 3 3 3 3 3 3 3::33:3 33 33 33 33 33:: 33 33 oomcaa' 33 33: 3 3: 32: 3 2: 3 3: 3 3 33: 3 3- 3 0: 3 2 32: 32: 33: 33 3333 mcaa4 S T D DF T L T IS~ L AE D V A rel. oomcaa~ 60 a C) 1 2 5 5 0) 0 0D j 0 0D 0) 0) C 0 0, pos occupied" 1 1 2: 2: 1 1 3: 2: 2 2 1 1 2 12: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 41): Analysis of V kappa subgroup 4 PCTIEP96/03647 I CDR III Lfl W. M- C> d 0 CN M~ un W M M) W M M M M amino acid' A1

B

C 33: D 1

E

F11 G 2: H 1 3: 2:

K

L 2 13 1 N 4::4: P 1:291 4 32:. R 2:1 S 2 23 2 1 T 2: 22: W 2: x Y33 3131: 29: 1 13: 15: 15: 15: 15S: 15: 3 unknown Q?) not sequenced 18: 18: 18: 18: 18: 18 sum of sq 00 mcaa' mcaa' 3 3: 331 3 3: 33i 33i 33i 33 33: 33: 33: 33: 15 33* 33 3133; 30: 32: 3 1; 29; 23; 22 91 VYY O Y Y S T P- 15:: 15: 15 15: 15: 15! 15: 15: 15! 15: 4! 00 000 rel.oomcaa' 00 CD C5 i. 00 S 0 :6 pos occupied' 1* 2 2: 2: 4: 6 7: 3! 31 11 11 1 1 1 8 SUBSTITUTE SHEET (RULE 26) WO 97108320 Table 4D: Analysis of V kappa subgroup 4 PCT/EP96/03647 _F Framework IV CO M CN v LO W(0 1 C sum amino acid'

A

D

E 14: F I G 15: 41 151

H

14; K 14: 13: L -4; M1

N

P1 S T 12: 14.

V 9: x

Y

unknown() notsequene 1 8: 18: 181 18: 18::18: 18: 18: 18: 18: 18: 18: 22 I1 183 68 154 105 82 228 6 135 158 258 27 136 195 264 116 499 236 196 69 254 106 518 sum of seq, IS:*.IiS' 15: 15: 15 15 15: 15: 15: 1S: 15.: 151 11 oomcaa& 12; 15: 15:: 11i 15i 14i 14; 9 1 1 5 3 1 mcaa' 1T F G~ Q~ T E I K rel. oomcaa' 0 80C: 0 6 0 0 Q0 r 0 pos occupied" 3: 1 1 2: 1 2 2 4 2 2 1 3 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 5A: Analysis of V lambda subgroup1 PCr/EP96/03647 I Framework I amino acid' -T 10 col M-.0 ~~U A j ~19: 8

B

C

E

G 22: 42: H 2: 1 14: L 1; 41 1

M

N

P 411 41: 1 41U 0 2: 1 41: 42: R S 3 9:1 41V J 41: T 41: 19: V V38 20: 1 1 42: x

Y

Z 16: 41: unknown P') [notsequenced 2 2: 1 1 1 1 1 1 1 1 1 111 0 o o.

.00.

0.

sum or seq' oomcaa' 40: 40: 41: 41 22! 39138: 41 41: 41: 41 41:: 41:: 41 41: 41i 411; 41V 41V 41i 42;: 421 421 421 421 41: 411 201 41: 22:: 20i 41:: 421 42;1 251 42: mcaa' I.Q IS IV L T Q V G AP GQ R V 0: C) C: 0: 0: C0 rel. oomcaa~ 2 C)i8 5 5 0 0 05 0 (n 0) Ch 0 0 0 pos occupied": 3: 2: 4i 1 1 1 1 1 1 1 4 1 3 4 1 1 5 1 SUBSTITUTE SHEET (RULE 26) WO 97108320 Table 5A: Analysis of V lambda subgroup 1 PCT/EP96,'03647 I CDRII amino acid' 0 r' L0 (Dj cO 00 LO o4 N C r4 MC A 2::1 2! 2!1

B

C 421 D ,331 3 1 G 42: 1V 2:39: 4: 2 H 2: 2: 2 1 41: 1 371..

K1 M1 N 21 37 13312 19: R 115 S1 42: 38: 3434:38: 13; 1l 1: 3 19 T 38! 3 4 3 2:1 7 2 V 1: 240: W 421 x Y 41 20* 7 z 36* unknown not sequenced[~-1 sum of seq' 42: 421 42* 42: 42- 42: 42: 42: 42; 42' 42: 42142. 42: 411 41 i41'i 411 42: a a oomcaa mcaa' 38: 411 42i 421 381 421 3 4: 381 371 7 39 131 311 3 6 20! 401 1IT 42 TI S~ C i S GS S S N I Gi GN N Y V S rel. oomfca a CoO pos occupied" 4: 2 1:1 C) C OiC 3146445 C: M

CO

CD

31 8~ 7~ 510 2 7i 1 I 2.2.a SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table SA: Analysis of V lambda subgroup 1 PCTIEP96/03647 a a Framework 11 amino acid' c. L. i n IL. Ln) i A 4::40:

B

C

D 131 0: 8: E 2; 5:1 K P 4214 SG 1 92 1 T 36: 1 1 8 1 Lw:3 :41 40 0 1 1 Ru fsq 4244244244244244242442 42 42 42: 42: 0 10.0 50 1: 1: o 0 0)0 W)~0 )0 -~C .os. cu ie 3 4 S~ 1 42. i5 9 3 x- SUSITT SHEET (RLE26 WO 97/08320 Table SA: Analysis of V lambda subgroup 1 PCT/EP96/03647 CDR 11 amino acid' l Ul UI LI n u-i WN Wl W WA W 1

B

D 38:

E

F 38: *G41: 2; 38 H1 I .17 K 38: L11

M

N

P 38: 38: R 14 2 4: 2 4 2 42 42.. 21 10 2 2 V 24 1 I x

Y

41: 41; 41. 41 42i 42: 42: unknown (Q) noeqenced 1 11 1 1 1 ~lJ3I V ac oomcaa-I incaa' rel. oomcaal pos occupied', 'HI: 'Ii 4 i1 1+1: 4 i 4I 41.: 41: 41: 41. 42 421 421 42: 421 42: 42: 421 38:1 40:1 41:1 41:: 411 411; T T 1 1 P S C>0 0 00) 0 0 3:t 2 00 C 0 1 42 411 241 38' 38: 42: 38; 42: 368 42; 38: 42: 4 2: C) i: CI)I 0 00 0t 0: CV:0 0000 1- 1 2: 3: 3 1 3: 1 3 1 SUBSITIIUTIE SHEET (RULE 26) WO 97108320 Table SA: Analysis of V lambda subgroup 1 PCT/EP96/03647 Framework Ill amino acid' r7 n n L o D Q 0 N 2 v L 6 A 13 41: 24 238

B

C

D 1 141: 37: E 1 2 4: f 42 1:

F

G 401 17: 1:42 H12 ~41~

K

L42: 41:

M

N1 P 2; Q 31! R 8: S 421 1142"1 2 4: 201 2 01 T 38: 18: 21: 17:3 W1 2 x unknown not seqce sum of seql 42 2 42! 42! 42' 42t 42: 42! 42i 42i 4 2 4 2 4 2 4 2 421 4 2; 42: 4 2; 4 2 oomcaa' 42. 40! 38i 42! 411 24 42: 24! 41 21 42: 41: 311 201 241 411 42i 38: 37! mcaa S G :Tj S A S LA I T G L QOS ED E A D re.oma, 0 0 a 0 r 0 ®r 0. 0- a, CO in 0: v CO:f)Q 2 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 5A: Analysis of V lambda subgroup 1 PCT/EP96/03647 CDR III M~ M C) C)04 M~0 0 amino acid' A 122 115:: 1 16:: 11

B

C 42: D 39:17: 7: E11 F 2 1 36: G 17.1 1 H11 K1 L. 1* 37:' 1*1 M1 N 21:2 91 P 161 Q 3 R S 4: 17:135 18 11 T -22: 11 1 V11 1 2 9 34: x Y 421:39' 3 1 3: 2: 4: 35: 39 38-- 38 1 unknown() not sequenced 1 1 11131 3 i31 31 33:333:343 sum of sea' 421 42" 42* 41: 41: 41* 41: 411 41: 41: 41: 41: 39: 391 38: 38: 39: 39: 36; to* 0 0 0 0 0 oomcaa' mcaa' rel.. oomcaal pos occupied': 42394 2 22122 38 39 17 35 37; 1817353938 38: 9 34; 36 Y Y CAT W DD S L S V VIF.

5 C- 1 cr V 1: Ln 5 :0 cn: Ln: -n C4:- 00 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCTIEP96/03647 Table 5A: Analysis of V lambda subgroup 1 Framework IV amino acid' a) 2 2 2 2 5? 0 u 0* 0O*e

A

F..

3 3631 36: 2 6

H

K 30: L 25: 34:

M

N1

P

03 1 1 18 R 12! S1 2.

T 1 361: 36; V 1. 36: 1 x

Y

z unknown (P) not sequencedi 4 61 6 6: 6: 6! 6! 6 6 10: 22 sum of 5eq' 36: 361 36: 36: 36: 361 36' 36: 36: 31: 19: oomcaa' 3 6: 3 1 3 6! 3 6 30: 2 53 6 36,: 3 4; 2 6,1 mcaa' G G G T.K L T V L G Q o: 0: C) rel. oomcaa. 0 80C CDD -11 :v n pos occupied': I i 4 1. 1 5: 1 1 3~4: 2: SUBSTITUTE SHEET (RULE 26) 285 84 224 81 87 559 188 141 344 176 296 251 156 720 359 282 92 202 16 524 141

S

WO 97/08320 Table SB3: Analysis of V lambda subgroup 2 PCTIEP96/03647 I Framework I amino acid' M~ Ln 0z co A 3 351 30: 6. :1

B

C

E

F

G 42; 42: H 21

K

L 40; 3:

M

N

P 42; 6 40 Q 221 4 1 41 V 42 R 61 1 S 141: 4 0: 42i 42i 43:' T 4 2:1 V. 12; 3 6: 14: x 1 Z 16: 42: unknown Q)1 gnot sequenced-3: 1 11 3' 1 1 1: 1 1 1 1 1 Ci"mof Coo~ An: AO: Al; Al) Al 1A~ 4 't 4. It 4. 4j I+J: 43: 43 !I r oomcaa' 2 241 35::4 04 24 1423040:42:36:42:42:42:40:42:42:43:28 mcaa' Q S A LTQ PA S- VS P Q rel. oomcaal a. a' 0' 0) C> 0) Ln 00 C) 0c -O CO O 0 -M C" pos occupied' 31 11 1 1: 3: 31: 21 2. 2 1 3 2 11 :.2 SUBSITIUTIE SHEET (RULE 26) WO 97/08320 Table 51B: Analysis of V lambda subgroup 2 PCT/EP96/03647

CORI

0 C nI nD C 0 <N tC1 Mt ct amino acid' (N N (N (N cN LU. CN 0-4 M (nMen A 3 11

B

C 42 1 D' 39 14: K 4 N 1 3: 4 1: 4328;' 4 1: r T 43:3:~3 V 37: 41 :4:3 8 14.2.

oT suenced9 1 31 su 37se: 44444444444 3333342424 ooca 434122639589332322444 T S T Y. x~, 0 reoma o 0 Y0 a 0 37lO 029:- n 0 .os.c..e 3 7 SUSTTUknHEowRUEn6 WO 97/08320 Table 513: Analysis of V lambda subgroup 2 PCT/EP96/03647 Framework 11 amino acid' Mw M'm.cr 0,.0 (f Ltn 0- c.CL n n A 14:

B

C

D 1 2: 201 21 E 20: 2: F 2T G 36: 2 H 2- 34 1 K 40; 41 1: 21: L 1 138: 6: M 26 1 N :2 1 8:12: P 41: 43: Q 41::39i 2 R 1U1 2 43: S12: 213- T 17 V1 3! 42t 39: x Y 41: 51 34: 2 p. unknown() 1 1 not sequnced sum of seq 2 00mcaa 1 inca a' rel. oomcaa- I 43.: 43;' 43;* 43 43. 43* 43! 43 43i 43: 43: 431 4 31 43: 43: 43: 43: 43: 43: 411 41: 39; 34. 41: 36: 40i 40431 41: 38: 2 614 3 34; 20: 39; 21: 21: 43: YQ0Q H P G KA P K L MI YD V S K R 10 &00 S:000 0' 0n 0n 0O 0 00: C)0o: pOs occupledr, 2 2:3 5 4: 2 1 2 4 4 2 3. 4.3 SUBSITUTE SHEET (RULE 26) WO 97/08320 Table SB3: Analysis of V lambda subgroup 2 PCT/EP96/03647 CDR 11 amino acid' tn 'Lo CO U w 0cc -n LCD C A 2

B

D 17: G43; 1 41: H 2: 3.

K 4 R 43: S 43: 2 8 43! 42i

T

x Y 2 431 4 3: 43: 43: 43: 43: 43: unknown P) not sequenced, a a a sum of seq' 41~ 43: 41~ 41 431 431 431 43 43: 43: 43: 43: 43; 43; 431 43: 43: 43; 431 oomcaa' 431 431 43: 43; 43 43: 43: 43: 39: 281 19: 43: 42: 43: 41: 42: 42; 43: 43: mcaa' P S G V SN R F S G tS K -eD 0 0:0,0- rel. oomcaa' C)o oo C)e~o 0: 0 0- 00 00 0 pos occupied": I 1 1 1 1 1 1 1 3 2 6 1 22 2 2 13' SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 5B: Analysis of V lambda subgroup 2 Framework IlI amino acid' r, l rr r -r O C OMC A 3: 14 31 1 3 6: 431

B

D 12: 3:42 39: E138: 43:

F.

G 3 9; 4 2: H 2: K1 L 431 431

M

N 38:1 p 2: 0~ 41 R 2: S 421 1 43: 42: T 1 41: 43: 1 2' x

Y

z unknown 1- not sequenced 1 sum or seq~ -%IIIa 4j: 4+j: 43: 'qj: 43 381 411 43;: 41. 43 43;: 35: 42:. 42: 43: 411 36: 38: 42: 43: 43: 39; T r mcaa' S G N T A S L Co. n C):C pusoccupied' 11 3; 4: 3: 1 1 1 T I S G L Q A: E D E A D CO0' M: V 0: 1 2' 21 2: 1 V 2: 4 4: 2 1.32- SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 5B3: Analysis of V lambda subgroup 2 CDR IlIl amino acid' Cw -co Q. S cc N. am CLO< Lu LL A 21 2 1: 11

B

C43: 11: D 31:2:1 E 11 F 3: 3 11 5: 42: G 1 2134 1 H1 K 3: M 1 N 575 1 P 14: Q 1 R 2 3:1 S 11 30:41: 1223.'.149: 1 T 164 4 j321 w x Y 4339 39 16 4: 1 36::42:43::43::43 unknown )2: not sequenced11 sum of seq' oomcaa' m caa' rel. oomcaa 5 POS occupied- 431: 431 431 431 42:: 431 43 43 43: 43: 42: 43i 431 431 431 431 431 421 42: 4 3: 39: 43: 30: 41: 39: 2 1: 21: 23: 14: 21 36: 42: 43: 43: 43: 11: 28: 42' Y Y IC S S Y A G S S T 1 3: 1: 3 2: 3: 7: 7. 8. 11: 6: 5: 2; V VF 1 1 13: 5: 1 133 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table SB3: Analysis of V lambda subgroup 2 PCT/EP96/03647 FwrmwoklV a) 0 v MN LLn (D CO.

amino acid' in 00 0 a CD 0> 0 0 0 Sum

A

B

D

F

G 4233:421 19,

H

K 36: L 2 8: 40:

M

N1

P

Q 114 R 11 2: 4: S1 2: T 7i 41 401 V 141 421 w x Y1 unknown P?) not sequenced 11 12 2 11 12158 sum of seq' 42i 42! 421 41: 411 42! 42i 4 21 4 f 25:' 14: oomcaa' 42: 331 4 2 41 i36i 28i 40 421 401 19; 14: MCaa 4 :G GOGT K L T VI GL rel. oomcaa' CO 00 C- CO to Q pos occupied':: 1: 4 1 1 5: 2; 3 1 2 3: 1 280 99 188 107 113 567 48 184 189 264 29 146 238 250 121 831 398 327 48 285 16 555 8 SUBSITIUTIE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table SC: Analysis of V lambda subgroup 3 Framework I amino acid' -4 tO LO n! A 1- 2: 2 7: 20:1: 27:: B D 5: E 20:1

H

K 2: L 37: 41 9:

N

P 2635: 1 2 7: o: 3 8 3 6: t

R.

13: 14! 1 1 281 37T 18: T 36 1 38: V 8 1 2: 3 4: 36: Y12314~.

-20: 38: unknown (P) not sequenced sum of seq' 38::3838::383838:38::38: 38: 38:' 38:;383838:38:38:38: 38:38: oomcaa mcaa' rel. oomcaal 20; 2 3: 201 3 7: 3 6' 3 8. 2 6: 351 2 8 381 34: 37: 36: 20: 27: 3 7: 36! 381 2 7; Y EL TQ P P S -VS V A P G Q T A rn )i C 1 -n C- i Ln- 0n o

CO:

pos occupied': 5! 2: 1: U 4: 31 4; 1 21 23: 21 41 2 2: 1: 3: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 5C: Analysis of V lambda subgroup 3 PCT/EP96/03647 (N N CN (N CN .N C-4 C l M Il M Cn Ml amino acid' A 15 1 21 3;

B

C 38: D 30::1 10: 3: 1 E 2: 2 1 36.-6 G 9:38 1 234: H 11 2 9 K 7: 2: 13: L 2 8: M11 N 2: 4::9:1 2:1 2: Q 10: 4 R 2 5: 2 10::1 S9 1 19: 10.12 8 1 T 3: 33! 1 1 4 V 3 20.

z 38: 38: 3 7: unknown(? not sequenced11 sum of seqI 38: 38 1381 3 8: 38: 3 8: 38:: 3 8 381 38: 381 38! 38: 37: 37: 37: 38 i 38 11 oomcaa' mcaa' 25! 38: 3.31 38: 19; 3 8: 30: 10: 38: 38 2 8: 231 1 1. 1-31-37. 20] 14 0RI T C G s S LG K-Y rel. oomcaa! pos occupied': 4:1 ao aL~0 CN n C V: Lf: C 315911359917 71C: Sj SUBSITIIUTE SHEET (RULE 26) WO 97/08320 Table 5C: Analysis of V lambda subgroup 3 PCT/EP96/03647 Framework 11 amino acid' CD Or) W M a N M~ Ln CDr CO M 0 N M v-

B

C

D 92228: E 11 3 3: F 3: 2:1 G 3 6;9: 2'.

H 1 31 1 281 K 321 2::6:1::13: L 2: 6. 6 33: 1:.

M1 1 N 1: 19: 9: P36 1 Q 137 351 136:1 9 1 R 1 4 2: 1: I38: 1 12: 1 14! 101 T 2 4 V1 1 31 4 3 7::91 x Y 3 5: 3 z S not sequenced__ sum of seq' oomcaa' inca a, rel. oomcaal pos occupied' 38. 38 38. 38. 48. 38. 381 381 38' 38 481 381 38: 38: J;J ~J 3 5: 371 351 322 361 36 i36 213 381 311,331 371,281 35: 91 22" 19: 13* 38: Y QQ KP G QA PV L VI Y D DNK R 0 0 C-4 Z A n: Co. 0 CO (N v 0 Co. 0) 0)4 i V)O D 2- 1 4:: 2 23-31 13 323131 7! 8. 71 91 V SUBSTIITUTE SHEET (RULE 28) WO 97/08320 Table 5C: Analysis of V lambda subgroup 3 PCT/EP96/03647 amino acid' Ltn O c 0 Lu O~ 0P Nc Low L LnLO Ln (D O D LO0 LO cn

B

D9 27 F 3 G 38. 38:

H

I .1 37:'

K

L

M

N 21: P 3 71 36:

Q

R 38 381 3:1 3 38: 12 T x

Y

z 38 8 3 383838: 38: unknown(?1 not sequenced11 1 Ce 4e ceo.

C*

C C

C.

Oe

C

C. CC em

C'

CCC CC

C

CCC...

C

CC..

C

C CeC p e e e

CCCC

C C Ce..

oomcaa 1 inca a.

rel. oomcaas Pos occupied" u. U. 37: 361 3.8 38* 38 P S 1 Jo:ad jai ji i 37 3.S 38 38! 138: 38! 38: 38 38.

38: 38. 38:: 37i 3 6: 2 7 38! 38! 38- 38. 38: 2 1. 38: 38: G P ERiF S G S c~i oi 0i 0 i 0 a 0: 0) 0 0 0 LA a iC 1111 21111 311 SUB STITE S HEET (RU LE 26) WO 97/08320 Table 5C: Analysis of V lambda subgroup 3 PCT/EP96103647 Framework III amino acid' W'T W' 7 r'mrZ n rDr-co a)CO "rnWd A 1~36~ Ii II1l WI 1342 38

B

C

D 38: 37: E -10: 14; 38: G 37: 28: H1 K1 L 38: 2: M N 2 8:1

P

Q 1 R 1:10 1 S371 2: 1 11: 23::1 T 1 6: 3 7: 25: 36: 12: 13: V 2 1 1411:1: x

Y

z unknown() Inot sequenced sum of seq, oomcaal incaa' rel. oomcaal 38: 3 8: 38: 381 38: 38: 38: 38: 38: 38: 38: 38: 38: 38: 38: 3 8 38: 38: 38; 37: 37: 28: 37: 36: 2 5: 381 3637* 23J 28l 14: 25S 34: 14: 38: 38l 38: 37l 2? i 0: 6: C C) CD~ OP 0 O n 6~ ~~0 pos occupied", 2 2: 5: 2: 21. 4j 1: 3: 21. 5i 2: 3* 5: 4: 6: 1: 1: 2 e SUBSITUTE SHEET (RULE 26) WO 97(08320 Table 5C: Analysis of V lambda subgroup 3 PCT/EP96,'03647 I CDR III amnino acid' Cw CO CO a) (nc OU "LL A 1332 12; 4:

B

C 3 8: D 321 6: E 12* 2: F 2: 2: G 3143 1 31 H 12; 1: K1 2 N 10 14: 112 2 1 0: 11 Q 2351 15: 31 36 373 no Se en d 1 1 14: 1 2 1 1 1 1 1 1 1 3 auill W l c oomcaa' m ca a4 rel. oomcaal pos occupied" 301 JO: 0: JO: .50i1 3 81 36:! 38:! 25; 14: 23 I 37i 3 .37.

132i 281261 37I: 36: 37:: 37: 37: 37: 37 14:: 10: 15: 31: 36: 37: 36 37!. 37 18; 28 a 0 1 Y YC Q S iW 1DSSGN.VV to i Co Cn co- O 0 r- O -~CO M: c.Or* v* Q 1 2 1 5 3 5 4 7 8 6 9 8 5 2 1 2 9 5: 31 15 4: 7 SUBSTITUTE SHEET (RULE 26) WO 97108320 PCT/EP96/03647 Table 5C: Analysis of V lambda subgroup 3 FrawrkV amino acid' c4 0000 'r csu

A

B

C

D

E 2:

F

G 35 31 3 5 1 i i 24; K 30: L 28: 33:

M

N

e- P1 0 7 R 2: S 2 T 4 35: 35: x

Y

unknow(? notsequenced 3: 3: 3: 3: 4: 3: 31 3 .4 28 265 82 225 145 461 32 160 110 233 17 126 249 275 154 501 347 308 62 211 603 1 89

S

sum of seq 1 oomcaa& mcaa' rel. oomcaa" 35 35 35: 31 35:: 3 5: 34:: 35: 35 3 5: 35: 30: 28; 35 3 5: 34: 27: 7: 3 5: 33: 24: 7: G GG T K L T V iLG Q 0 p a- 0 0 all00 0 0 C 0 C0 0 a SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6A: Analysis of V heavy chain subgroup 1 A PCT/EP96/03647 I Framework I amino acid' 04 r1a (D u0 (n C C A1 14; 60: 24: 1:

B

C

E 1 2 1 2. 64:

F.

v H 2: 2: K 2 5764:6 L 2::59: 3:

M

N 6: P 63: S 53. R 13% 3: 1 40:63; T1 V 2:55: 155: 61- 64: 64: x r- unknown() not sequenced 11 10: 10: 10: 10: 10: 10: 10: 6 6: 6: 6: 6 6: 6: 6: 6; 6: 6: 6: sum of seqI 59; 60: 60: 60::60. 60: 60: 60: 64; 64: 64: 64: 64: 64: 64; 64: 64: 64: 64: 64: oomcaa' nca a' rel. oomcaal 53::55! 56: 59; 55; 4 5: 60: 58::60: 641:61: 57: 64: 63- 64i40i 63i64: 60:64: Q V Q L V Q S G A EVK K P G S S V KyV 2? i g 0C 0 in C:0i 0M~l T..9.LM. C J *l n: CO 0 mj0 O 0 pos occupied' 4: 4: 3: 2: 4: 31 1: 2 3: 1. 2: 3: 1: H 2 2: 1 3: 1: I 2-- SUBSiTUTIE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 6A: Analysis of V heavy chain subgroup 1A F

CDRI

amino acid' Z CN. CN c--4 C4~ Cq c.q c-i rqCn A 62:

B

C 63: D 1V

E

G 1 6941 23:~ K1: 63 1 1 2 M 4i N 2$5 4: P

Q

I. S 63: 68 1 4060 *T 12; 681 25::3: *V 169: W 701 Y 27; 64: z unknown Q) not sequenced 6: 6: 6 5: 2: 11 sum of seq 1 64: 64i E64 651 68: 69; 70: 70: 70:: 7Oi7 0* 70: 70: 70: 70: 701 70' 70; 70; oomcaa 3 631 631 63: 621 68: 69; 41' 68: 69: 401601 701 70; 64: 4 1: 61: 60: 70: 69170: mcaa' SC KAS G GT F SS Y A ISWV R rel. oomca i a-~ pos occupied': 2: 2 2; 3 1 1: 4 3; 2: 6: 5 1 1: 4 6: 4 5: 1 2: 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 6A: Analysis of V heavy chain subgroup 1A Framework It amino acd PN m 4 nwr o( L

C

D1 F. 2 339: G 1:68: 69 1 69:39 1 68: H1 65S;3 8 34:

K

L 1: 68: 1; 1 1 24: M 67: 21 4 N 4: 32 2: 1 1 1 1 R 68 1 1 4 4: 69 V 12 1: 1 2 R 4* 1 20 41 zV 70.. unnw no sequenced su ofsq200000007777 00000 0007 I~noo sequenced ofeo. sI7i7i7i7: 0:7: 0 0 0 0:0:7: 0 0 0 0 70 0a0 0

U

Aoocuid 21 3 2 24 2 4 5 11063EW' GG IIP SUSITT HET(UL 6 WO 97/08320 PCT/EP96/03647 Table 6A: Analysis of V heavy chain subgroup IJA a in ac d' L. OW QD (It r-Lno cD r 0) cN' crn r''Ln A H34: 3:;

C

D 15: 1 2; E 133: F 148: 3: 4 67 H1 14: V144:1 K 1 4 7 1:1 *2 M 21; N 9: 59: 18: P 1 7: Q1170: 64: R 2: 2 1: 69 1 1: 12 1 570' T 3 4 26: 3: 66: 65:.24: ~27: ~67 V 11 65 S 3 V. 1 65. 3 w x z unknown not sequenced sum of seq 70: 70 U70 70i 70i 70: 70: 701 7~ :10101.70101W7 oomcaa, mcaa' rel. oomcaa- 341 34: 5 9 68 69: 70: 47. 48 64 67 69; 65: 66: 44: 65 4:70330 7: T A N Y A 0 K F QG R V T I TAD ES T C" I W: M: a: 0:: 0 0 osO: (n :e o C01 (CD pos occupied": i 6: 7: 31 2: 1 4 21 5! 3 2 3 3 4~ 2 3 1~ 5.1 2 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6A: Analysis of V heavy chain subgroup 1A PCTIEP96/03647 Framework IIl amino acid' 'r QZ'r" o 0 3 (N L(n WDI O0 00a A 64: 1:

B

C D 2: 26:70: E 64: 44: F 1 2: 1 21 K 3: L 3: 63. 7 0: 2: M 67 1 4

P

R 3 1231 6 S 62: 1 41 :j49; 67 1 T 1692: 11 32: 4: 67; V 3 4 164: x Y 168:-. 69:68: z unknownP~) nt sequenced sum of seql 70:*70 7070110707070:7070:70:70:70!70170:70:70:70:7070:

S

oomcaa' inca a, rel. oomcaa-1 occupied,: 62 :69: 64: 68: 67: 64 63: 41: 49: 701 621 67:! 44170 67 70- 64: 69 68: S T A YME L5 S ILR S EQI TA V Y YC 0 a-o a ae 0 a-.o 0~ a..:0 0)~-fs f~0a~000Cn (D 00 CD a) 0 o~ C .O 4 2 43::2431:6::61 4:221 12 I L SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6A: Analysis of V heavy chain subgroup 1lA PCT/EP96/03647 1 -4 Ln r 00 C) CDR IlI amino acid' M0 nC n -0N GG 2: 1. 4: 1: 2 2 1! 1: 1: 1: 11 21 1

B

C 11162 11: 721 D 165 3 35 434: 1: 1:14: 591 E 9 211 1 F 1 3 2 3 1 2 2 1 281 2: G 21 141 13: 2 0: 101 141 5! 20i 15: 16" 3 3: 4 15: 1 1 7:: 2::52:2 221 1 1 K L 144 25;2:11 42 11 N 221 12 2 114 P 2 23 1 3 12224 2 1 1 Q 2 1 3i 1 31 112;2:4 2 l 4 U 1 1 R 155: 11 51 7 81 1 41 2! 1 1 16? 5 11 55 521:511:8 4 3 21 2 W 113 1:1 2 3: 15:1: x Y 1: 2: 3 20: 5: 4 9.1: 2" 11: 20: 10i 6: 9 10: 7 1 z 1: 2: 2: 3 6:11: 11!114* 23! 26! 2 6! 31! 34: 46' 39:21: unknown 1 H 2: 3: 7 Lotsequenced 2 2:24';41 4!:4 St 5 5 5s: 5 55 sum of seq' oomcaal mcaa' rel. oomcaa, pos occupied": 6655:1-6:20: 20' 20: 16: 21: 201 15;:161 231261 26::31134! 461 39: 2 8: 59: FD. cn 0. ;0 3 8: 101 14: 18: 151 18: 15: 15 17: 17T 151 121 Ill 1 101 81 7 61 6 U SUB STITUTE S HEET (RU LE 26) WO 97/09320 WO 9708320PCT/EP96/03647 Table 6A: Analysis of V heavy chain subgroup 1A Framework IV amino acid' 0 0 00> su m

B

C

D 1 E 1 *F 2: G 58: 59: 1: 1: H1 K 1: L 3: 4 40: 1 M 13: N1 0 52* S53: 51 T 5 4111: 1 51:: V 15: 1 154; 54: W 59: 1 x Y 34: 1: z unknown Q?) not sequenced 5: 9: 9 10: 11: 141 14114: 15: 16: 16: 17 670 165 308 297 226 928 14 286 325 386 189 176 238 494 351 972 736 '699 243 542 3 578 8 406 sum of seq' oomcaa' mcaa' rel. oomcaa-" pos occupied' 65: 61:61:60759 34: 59: 58: 52: 59 Y W; Q: 6 54i 54: 53 54:::531 51: V S S 9: 3 4 1~ 3: 3: 2: 1 3 SUBSTITUTE SHEET (RULE 26) WO 97109320 PCT/EP96103647 Table 61B: Analysis of V heavy chain subgroup 1 B I Framework I amino acid' cq n-,t Lf W r- c n 0 M U T 0 e-2 a) T 0

B

D

E 15: 1:

F

27.

H11 K 31 3 4:331 33: L 3 26: 1 M 1

N

P 133 1 Q 2 1: S27 1:34! T 112: SV 3:21 201 IS 35 3 4: x

Y

unknown P)J not sequenced 15: 15: 15' 13; 13: 13: 13: 13: 6: 5: 5; 5: 5: 5: 5 5: 5: 5: sum of seq' 25::252 52 72 7;:2 727:27:34:35:35:35* 35353 535:35::35:35:35: oomcaa incaa rel. oomcaa, pos occupied" 21: 21: 20: 261 20' 26: 27: 27: 321 35135: 34: 33.:33. 35::34: 34; 351 33: 34: .V o L V Q S G A EVVK K P G A S V KyV CO a)O .i:a M t::a a) )n 31 31: 41: 21: 41 21 1 3: 1 i 2: 2: 3: 11 21 2 1 2' 2: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6B3: Analysis of V heavy chain subgroup 1 B PCTI/EP96/03647

CDRII

amino acid' C' C 2 1, 2 C '1 3:"11c Nccnz MMM

MM

A 3 1 121 6 C 3 D E ,31 G 1 40: 1114:1 H 31 34: 1 1 9: M 23: N 1 3 13: P1 .1.1 R 2: 2:1 37: S 351 40 51 2;:15 2::1 T 31 3 2 1 13411 V1 1 12238 W x T- Y 36 1: I 32:19 1 z unknown (Q) not sequenced, 5 5: 5 sum of seq' 35: 35135. 35: 40: 40: 40: 40: 40* 40 4040: 40:40: 40: 40: 40: 40: 40: 0W

S.

5S

S

S

*5*S

S

S

S

oomcaa' m ca a rel. oomcaa' pos occupied' 353 52830404036:32:39:341 5 40:40:32119:23:34140:38:37 A V R .4 2~ 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 683: Analysis of V heavy chain subgroup 1 B PCT/EP96/03647 Framework 11 T amino acid' .4 o t -c n 0 7;S4< c n ',L fees of :4069 a

S

S

p 5SSOS

C

S

*~S

S

Q A P G Q G 0* C. n C O e M CO: ):Cor L E M0 O 40 40: 40: 40; 40: 40 40 40: 40; 40; 40. 3 7: 3 9: 33 34 3 5: 31: 40: 40: 20: 2 0; 39: a- a? a 2? 00 0 rel. oomc22 S pos occupied". 2: 2: 2: 2: 1 2~ U 21: 2: 4: 41 V 9: 8 2: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6B: Analysis of V heavy chain subgroup IlB PCT/EP96/03647 CDR 11 amino acid' wL Ln L" to W T_ Wm_ -7 q A 1 21 271 211 21

B

C

F 1 41 39 H 2 1 11 1. 1 13 .2 4K 228 36 1 L 15: 1 14 1: R: 23 1223:3 S 21 211 1: 1 37: T *35* N 11 3818 W 3: z 4 unknow noVeune sum.. 40 40480 0 4 04 040 4 04 0 04 04 183 363 93:43 83 2 4 3 3 3 72 .TY Q F G V M R T SUSIUESEE RL 6 WO 97/08320 Table 613: Analysis of V heavy chain subgroup IlB PCT/EP96/03647 Framework III r r M ;0 CO W) W W 0 W W (Nr amino acid' A 35: 1 2: 4 0

B

C 37 19: 401940: E -35: 19: F 12 21

H

K L 2: 3 9: 39 2: 1 M37: 1 2: N 7 12: P 1 .1 0 4 R 41 216: 37: 2 271 1 35:20 1 3 6:1 T 113911 V 4: 1 1 33: x Y 39: 3 8;35; t I. unknown no2.t sequenced 1 1

S.

S.

555555

S

S

*5*5 sumi u se oomcaa 3 4ui Q:40. 40 4U 40 40~ 40 401 40l 39~ 39 39 39 27j: 391 35: 39i 37! 35i 391 351 20: 39: 37: 36: 191 40: 40: 40: 33: 38: 35: 37 mcaa, Y M: E L rel. oomcaal 2? a pos occupied" 51 2: 3: 21 3: 3 2 SS RS 0 AV Y YC o 0 0 00 0~- CO 0 i O r0 00 C, C 0 5424431115243):a):Cn SUBSTITUTE S HEET (RULE 26) WO 97/08320 Table 68: Analysis of V heavy chain subgroup 1 B PCT/EP96/03647 L CDR III amino acid' 2 M M0< cMD Mu' 2. c-i- c0 0 A 3 7i i 6~ 1 2! 3: 1: 3:

B

C13: 21 D 7 5 231:5:4: 1 22 1:2 27 F 1: 1 3 1 1 12 121 F 3:.1 75 94 1 221 2131: H 1 2!7 5: 9 7 1:1 :2 M 221 1 N: 11 11 11 1 31 2. 4 I T 212 21 11 141 unnw 1?j 315:1 1 no Seun d 1 1 3 3 3 3: 3 3: 4 4: 42 4 4 4 4 a sum of seq' oomcaa 1 mcaa' rel. oomcaal 371 37i 37;. 37:: 37:: 37! 36: 3613 36 3 3 6 3 6: 3 6: 3 6: 3 6: 3 G: 3 6 3 61 32?: 31: 71 71 5! 5: 9 81 0:11: 14: 20; 23:25: 25: 25: 23: 18: 15 27; A R D G D G 0D go 00 go: 2? ao &0ie i2 cj) c.sj co: m: q. m (0(0 cJ) t j PuSOu'.x uu S 10J: 12:: 18 131 13!-12: 2 17: 14: 13:10: 9: 8 8 8: 5 SUBSTITUE SHEET (RULE 26) WO 97/08320 Table 6B3: Analysis of V heavy chain subgroup 1 B PCT/EP96/03647 Framework

IVI

amino acid' 00020 OO2

A

B

C

D 2:

E

F 1.

G 127 T 126 H1 K 2: L 12: M 2: P1 1 Q 23: R1 S 31 18::18 T21: 6 16" 1 V 612 18: W 29: x -3: unknown Q) nt sequencedl 4: 11 13: 13: 14' 19: 19: 19: 20: 20: 21: 22 sum of seql 3 6: 29: 2 7: 27-* 26: 211 21; 21: 20" 20: 19; 18: sum 340 79 179 159 130 450 51 113 194 204 144 138 128 253 247 432 390 342 158 294 394 3 458 oomcaa' I1 3:29:27 23 26: 21 12: 21: 16' 18; 18: 18: mcaa' rel. oomcaal pos occupied' Y W:G 10! 11 Q GT LIV T V IS S o ea 60 Oi Ooi 0 4 411413321 SUBSTITUTE SHEET (RULE 26) WO 97(08320 Table 6C: Analysis of V heavy chain subgroup 2 PCTIEP96/03647 Framework I amino acid' t LO cD r- 0O A. 3 E 16 2:

F

H

K 3 6: 1 L 6: 6: 6: 6

M

N1 P 1 6 6:1 R 2 T 6 1 2 S V S 6 w x

Y

unknown P~) notsequenced 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 sum of seq 1 0omcaa, inca a' 66 61:6:6 6: 6: 6: 6 6. 6 6~ 6: 6: 6: 6 61: 6 6: 6: Z V~ T1 L K E S G P1 Al L V K P T Q T 1 LI TI L rel. oomcaa' a- o:deC 0000 00 0 0 D c 0 00: 0-r. CD 0 C0: pos occupied' 3 2: 11 1 3 1 l 3 1 i 1 3* 1 1 1 1: 2. 2 2. 1:1 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6C: Analysis of V heavy chain subgroup 2 PCT/EP96/03647

CDRI

amino acid' ;7 L4w c.CNM <MMC 1 C 7: 2; D1

E

F 3

H

1 7:

K

L 2:6: M 51 N 2:

P

R 2 1 7: S 1 6: 6: 4: V 2 2 7: W 7: x Y1 z unknown not sequenced1 sum of seq' oomcaa 3 mcaa& rel. oomcaal bi 7: 7: 7: 71 71i 71 7 71 7: 71 71 7: 7: 7: 7 V C T F S G F S L I S SI G M G V CD a 0 6 H 1 2: 2: 2: 2: 2: 3: 41 31 21 4; 1 :S W I R -0 100 D pos occupied"-':, 1 SUBSITUTE SHEET(RULE 26) WO 97108320 Table 6C: Analysis of V heavy chain subgroup 2 PCT/EP96/03647 Framework 11 amino acid' 't Lnwr'c n0L

B

C

K 6: 7 1 7: 2 P 5. 7

V

W 7 1 x11 Y1 1 4. z 6:7* 7: 0 unknow() not sequence*d

III

sum of seql 7 7: 7: 7: 7 7: 7: 7 7 7 7 7: 7: 7: 7: 7 7: 7: 7 7: 0:0: oomca 6 776677 777 6 6 7 3 mcaa' Q P P GKA L EWL A H ID DO0 rel. oomcaa:~~ a 0 0 Oo-.0. OOCCO 0. S R0..0 0: 0 poS occupied' 1 1 221 V I: 14 5 11 3 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table GC: Analysis of V heavy chain subgroup 2 PCT/EP96/03647 CDR 11 amino acid' Lw t' L o W4m

A

B

C

D 5: 61 F1 1 H1 K 1;61 4: 6: 6: L 7 7:

M.

N

P 2: R 2:1 21 71 1 6 7: 4:1 2: 6- T: 7 V1 x1 Y 3 4: z unknown(? not sequenced sum of seqI 71 7 7: 7; 7: 7: 7: 7: 7 7 7: 7: 71 7: 7: 7 7 7: 7 oomcaa 5: 6' 3 4; 6: 4 7 7 4V47 6 6: 5 6: 6 67 mcaa' D K Y Y S 1T S IL K S R L T I S K D T S K o o.D 0 0 Q rel.~~ oomaa a,0 c f fO cc i o pos occuPIed' 3 2: 3 4; 2: 3 1 V1 3: 2: 1 1 2: 2: 2 2: 2. 2: u 2 SUBSITUTE SHEET (RULE 26) WO 97/08320.

Table 6C: Analysis of V heavy chain subgroup 2 PCT/EP96/03647 Framework III amino acid' 'r 0W E.c oUL (D co- 3c A1

B

C 7 D 71

F

G 2

H

2 1

K

L 6: M 7: N 5: 6:.

P7 Q 7: R S 2: T 5: 5: 7 7; V 7."7 6: x 7 z unknown(? not sequenced .4.

a 4.0 .4.0000 4.

*0*a 4.

*00000 sum of seql oomcaa'1 incaa 4 rel. oomcaa' pos occupied' 7 7i 7i 7: 7: 7 7: 7: 7 7: 7 7: 7: 7 5 7: 7: 7 6: 5: 7: 5: 6: 5: 6: 7 6: 7 7: 5 7: 7 7 7! a'a'2 C)0 0 101 2111 0:0 a 00 COD W. 0 -*CO0 2::21 132::32:1 21121:;11 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6C: Analysis of V heavy chain subgroup 2 PCT/EP96/03647 CDR III (J M UI U..0 I a mino a c id' A 5 121

B

D 6 1 F3 G. 1 1 2 1 1 H1 1 K1 L1 1 2 V 2 1. Y 2221 1 0 z 22 3: 4 4 4: 6: 5: 3: unknown Q?) no.t sequenced 1 1 1: 1: 1 1 1 1 1 1 .0 'S *SScS* sum or eqoomcaal inca a' rel. oomcaal pos occupied", 0: 0. b. b: b: b 6! 6i b: b b: 6 6 6 5: 6: 3: 11 2: 2; 1; 2' 21 21 2:i 31 4: 4: 4: 61 5 3: 3 6 A R I H NI GE A. F FD 2e i i -0 0 0 -Q 0 0 c, tLn:( C.D CO: Lfl 'e7 SUBSITIUTE SHEET (RULE 26) WO 97/08320 Table GC: Analysis of V heavy chain subgroup 2 PCTIEP96/03647 F Framework IV amino acid' 00 0 0 c)c su m A1

F

G 6: 61

H

K11 L 13:

M

N

P31 1 V: 33 W 6: 6 Y1 unknown Q~) notsequenced 1 1: 1: 1: 4 sum of seq' 6: 6: 6: 6 6 6 6: 6 6! 6 6: 3 oomcaa 3 6' 6 3 6: 6 3 6 5: 6: 6: 3 mca VWGQGT L:V T VS S rel. oomcaal C-o) 0: O: 0 0 00 i0: pos occupied" 4: 1 1 3 1: 4 1 2 1 0 0 0 0 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6D: Analysis of V heavy chain subgroup 3 PCT/EP96/03647 I ~Frameamino acid' cN rr LO ID co C N Cn~ A 1112:1: 1 1. 1 C. 110: 9151166: q:8 2: F41 G181: 193: 174 1 202: H 54: 3 26: L 1 5: 176: 43: 140:1 1194 Q 41: 138 1: 3: 12: 162: R 6: S178: 2. 8 T1 V 5::1471 1 118 62: 195::

Y

Z 8.

unknown (P) not sequenced 47: 47: 45: 33: 32: 3232; 31: 10; 7 6: 6: 6: 6: 6: 4 sum of seq' oomcaa3 inca a' rel. oomcaal pos occupied" 165: 1G5:: 167; 1791 180* 180; 180: 181: 202: 205: 206;: 2061 206: 206: 206 0: 147: 1381 1761 118:: 166 178: 181: 193: 174: 140: 195: 162; 194: 202 V Q L V E S G GG G I V Q P G en CO Oj CO -h o C O..

5 4: 7: 4 4 -1 11 A A SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 61D: Analysis of V heavy chain subgroup 3 work I amino acid' CO (Nj rn CId'C~4C~jCO (n A 183: 192: 1 C 1 209: D 7: E 8:8 F 1 11201: 2011 G 134: 2. 207-; 3

H

3 17 i K 154 L 205: 201:6 31 M11 N 110: P 0 1: R 62: 191 11; S206: 207: 4: 2!:209: 15: 174: T 4: 1 2: 4: 4 1 163: V 8: 7: 9i 1 6 x

Y

unknown() not sequenced 4 4 4: 4 3 3 3 3: 3 3 1 1 2 1 2 sum of seqI oomcaal mcaa' 208.: 208: 2081 209:: 209 209.: 209: 209; 209: 211: 211 210: 211 210: 206::205 S L 191:: 201: 207 R L S rel. oomcaa' pos occupied" 4 3: 4: 3 2: 3 209: 183; 192: 209; 207: 201: 163; 201 C A A S GF T F o o C- co 0 n L n 4 5a 174:

S

5: :3 4 81 41 7 SUBSITIUTE SHEET (RULE 26) WO 97/08320 Table 6D): Analysis of V heavy chain subgroup 3 PCTIEP96/03647 amino acid' c c' Cn M en. 0 I-It t t A 11780: 1 1 18

B

C1 0 268 3: 7 2! E 110:

F.

G13: 31 1 209: H 4 8 8: 1* 1* 15: 12: K 7T1 20 2: L 3 3! 2 31 2 1 M 193: N 35 8 3 34.

P 114 191: Q 209: 1 R 7: 207: 7 8 S 103 1 7j 8: 7 2: 314: T9 15: 10: 4 V 2: 7: 1: 197: 2: W 30: 212- Y 1154: 19; 3 z -210: 210: unknown

M?

not sequne 2: 2: 2 1 1 sum of 5eq~ oomcaa' nca a' rel. oomcaa' pos occupied", 210: 210: 210: 210: 2 101 2121 212: 212: 211; 211:: 211:; 212 212:: 212:: 212:: 103 2102 210: 801.193 88 212: 197; 207: 209: 187:: 191: 209: 202: SY A M H W V R Q A P G K M CO Cn 00-0 (n Ch 14 1 1 9 10: 4:9 1 3 3 3 9 5 4 4 SUBSTiTUTE SHEET (RULE 26) WO 97/08320 Table 6D): Analysis of V heavy chain subgroup 3 PCT/EP96/03647 Nvork 11 amino acid' V -IT L nU n L A 1;77 42: 11 2: 14: 7: D 17: 9 483: E 198: 3: 2 1 2 1 F 7: 1 2 1 1 8: G 207: 3 3: 11 10: 46: 4:163: H 6 1 3::191:1 K .1 37; 2: 30: 3: 1 L 211 5: 12: 1 M1 1 N 13: 7 9: 2 13: 11 1 Q 710.

R 124: 1 17: 5 1 2: 16G: S3: 1 0 1 9 118: 432 1 74: 17: 82: 3 5: 4 2: 13.!12: 3: V 3 204: 49 2 1 6 W 210: 1 86 X 4; 3: 12 Si58: 8 unknown P) not sequenced sum of seqI 212: 212: 212. 212: 212: 212: 212: 212: 212: 212: 212: 212:: 212: 212: 212:

S

S

S

.5 4

S

S

oomcaa, inca a' rel. oomcaa' 207: 211: 198: 210: 204: 102: 49: 191: 118B: 58: 178: 178: 94: 163: G L E W V S V I S Y D G G ac, a' 60 all 60 SOcD aO) M a) I- O Ln (N~1~ pOS OCCupIeO 4. 2. 3:1 3 3~ 15: 9: 11: 19: 5: 5 12: 9: 12; SUBSTITUTE SHEET (RULE 26) WO 97/08320.

Table GD: Analysis of V heavy chain subgroup 3 PCT/EP96/03647 @9 9 99.* @9 9@**99 9 CDR 11 amino acid' 'Lo [n L0 '~'CDC0 t O L I 0 w L A 9: 1 2: 174133: B 1 2:

C

D 1 1 17: 1601 E 8::3 2.1 2: F 13 2: 207: G S 5: 212: 1 H 1 4: 1 3: 3 7 2: 8 14::2081 K 1 611 199: 8: M 8: 2: 1 N 51 4: 2 2 P 1 16: 818: 1: Q 32: 21 2: R 5: 4. 5t 6: 201: S 481 11 4: 193: 2' 7 211: T 429751 7 1 89i V 2 102 204 1 3 W 2 X 4; 1 Y9 151:210'11 z unknown (M no2t sequenced MCaa4 rel. oomcaal ZI 14:~ 4 I 14: 4I~ 1 Z4I4 44 L14: 414' 414 LIL Z:4 LIZ 51: 9V~ 151: 210:: 174:; 1601 193; 204~ 199: 212 2011 207 LIZ! 414: 1-14: 189: 2081 211: N T Y Y A D S V K G R F T I S n C-4 n L* 00 ui CO: 0 C'4 v- cm; 00 r o pos occup edr, IT: 12: 15 2 8: 3 2: U 4: 5: 5; 3 2: SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 6D): Analysis of V heavy chain subgroup 3 Framework III amino acid' ;7 r' r r' .O mr OC A I57: 1 81 B D 199381 22 1 E 6: 4: G 141 .188:209:..1.21 N: 25 173:8 3 1 P 18: 3 R 211 109 1: 28 12 15 81056 6186.. 0: 3 2 T 142 170 2:48:3: 11 0 V 11 R 221 4: 1 z 5 8: 1 1 56 :1 6 u n k n o wn. 7) n T squnce 1: 1 9.

S 9 *9 9 99 99 9* 9 9* 99 9

S

99*99* 9 9 sum of seq' oomcaa 1 incaa' rel. oomcaa 212: 212: 211: 211: 212: 212: 212: 2121 212: 212::212: 212: 212: 212 212: 211: 199: 170: 153: 186: 188; 1421 1881 194 209: 199: 205: 181: 186: 212; R ODN S K N TIL Y L 0 MN S L 01 0: e: o0 a o: a Ln C :o 0O~ en: CD t C,4 a) .r f pos occupied 4: 1 7: 6: 5 5: 3. 6. 4. 11: 7: 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table GD: Analysis of V heavy chain subgroup 3 PCT/EP96/03647 amino acid' 2Q a' 0'0 0' a 0 2 C) C a) C" a) M A .149: 1i 12071:7: :1 9 1 C 1 210: 5; 2: 1 D 5: 15::209: 21 54: 7: 6 1. 19 G1 1 61 4: 1 28134126:35: H 1 1:311: 8 2: 4: 15: K 301 60: 4: 3 L 181 611: 7 M2:1 6: 1 N 1: 12043: P. 91 1 3 429:10: 1 51 3 91 2 R 177: 103 9: 30: 19: S 113 9 8:11i T 3 281 2071 1 25157: 6!20: V 9: 187 10 1 7 7 x.

Y .211: 194' 12: 9 8!; z unknown Q) not sequenced 1 111 1 1 1 1 7; 12: 13; sum of seqI 212: 2121 212: 212! 211: 2111 211: 211: 211- 211::211: 211: 205: 20 199: 6O 0 0 0@SO 0e 00 S

S@

OS

S S 0@ OS 0 S 0 S S 0

OSS*SS

S

5@55 0 oomcaa 1 inca a' rel. oomcaal pos occupied' 177: 1491 190: 209: 207! 207' 187 2111 194" 210: 173: 103: 54: 30: a- 0 a a 10 4o 4 4 2o 7 1O 4o 2 4 8 20 2 SUBSITUT SHE (RUL C6) WO 97108320 Table 6D): Analysis of V heavy chain subgroup 3 PCTIEP96103647 CDR IIl CD) 0D 0 inC) LU 0 amino acid' A 713 7: 9: 6: 2: 5: 5: 9 13: 2 C 13.: 5: 1 2: 11 3: 2:1 D 11 7::104::2 3103: 3 1 32: 146 E 6: 3: 1 13: 1 11 F. 3: 54 5: 5 63: 5: 7 2: 1 165 1: G 34173 5::17T1412 3:10: 5: 1 .53232 3: 6 H 34 3 2 9 2 1 31 1 2 8 1 6I 611 3: 1 310: 3: 3 2: 1 2: K 2::11 3: 1 L 2613:: 412 310: 3 2: 1 M 1: 2 1 32: N 464322:6; 25: 2 P 655 698;2 3: 2: 1 3: 9: Q 41 1 1 1 1 11 R 4109::7: 5: 52: 3 1 1 2; 4: S 16: 28: 2 7: 25: 24: 8 11; 9: 3 2: 3: 1 1 1 T 6 12 9::171 7: 1 2: 5: 11 1 V 13 715436 212:1:1 1 W 6 5: 6: 7: 2: 4 16: x11 Y16: 14:1 5: 8: 18: 20: 13: 20; 25; 28: 32; 28: -12: 2 1: 35: 5 4' 73: 8 7 102 110: 126: 135: 134: 120: 91: 71: 2 1 unknown 2: 1 1 3 2 not sequenced 14: 14: 14: 14: 15 19: 21: 22: 23: 23: 23 25; 25: 26: sum of seq' oomcaa 3 1 mcaa' rel. oomcaas pos occupied' 198; 198:: 198 197: 196:: 192: 190: 189: 188:: 188:: 188: 186 186: 185: 186: 3 4: 28:3 4 7 87: 102: 110: 126: 135: 134: 120: 91:7 4 G S IG

D

20: 20: 19: 20: 19: 20: 17! 14: 14; 12: 12:. 1 121 111 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96103647 Table 6D: Analysis of V heavy chain subgroup 3 Framework IV amino acid' D 0 0 sum A 121 1767 B 13 C 470 D 2: 1121 E 1832 -F 2: 807 -G .140: 1 1301 1: 2743.

H 4: 179 1 15;1 651 K 13 933 L 10 1 91: 2 1881 M 6: 496 844... P 17:1 1 568 Q 11' .949 R8 1413 S 7 1 118. 110 3009 T 123: 27: 122 i 1 1426 V 34: 1 1 1251 119: 1851 .W1581 686 X 26 Y 82 1598 z 8 2: 2 2: 2' 2: 2: 2 2 2 1 112023 unknown ()12 not sequenced 27: 50: 67 7 8 1 8 84 8 8 9 9711650 sum of seq' oomcaa mcaa, rel. oomcaak 184:: 1611 1441: 136! 1331 130: 128: 127: 125: 122: 119; 1141 82: 158: 1401 1111; 130:: 1231 911 125- 122: 119: 118: 110; 6o 10 al al 0r s~ LnA co r. c,4 i co tn co co c a pos occupied': 12. 3i 4; 6: 3 6: 1h 1 4 SUBSTITUTE SHEET (RULE 26) WO 97/08320 PCT/EP96103647 Table 6E: Analysis of V heavy chain subgroup 4 F Framework I amino acid' -NM-z wn C L2 ON A 19 11 44...

F.

C 9 1 54: 1::51 2: H 4 2; 54 1..

L 7: 5 4 53:19: 1: 53

M

N

P 33: 511 4' Q 52: 50: 51 2 0::17 R1 S33: 52! 52: T 152: W

Y

I.

unknown (Q) not-sequenced 3: 3: 3: 3! 4: 4: 4 33 4 4 3; 3: 4: 4 4 4 4: 3: o sum of seql 154. 54! 54: 54: 53; 531531 54:154. 5 3. 5 3 oomcaa' 52; 47: 50: 5 4: 51: 32: 33 54: 33: 53: 53 mcaa 4 Q: V Q E S G P:G L rel. oomcaa~ so COD i: ao S:Q M0.0. cc0 pos occupied': 1' 2~ 2! v 2. 3 2 i~4~ 54: 54: 53i 53: 53: 53: 53: 54: 53: 34. 54: 51: 52. 44, 52: 53. 521 V K P !E I T L .S L aI 0 Zo 3 1 2* 3I 2 3 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6E: Analysis of V heavy chain subgroup -4 PCT/EP96/03647

CORII

amino acid' eJ CN QN C'1 CN c.0 CO. CO M M~ M~ M A

B

C 53 1 D 1 41:11: 1: G 53:5 3 112 1 3 41 8.

H 12: 1 132: 51

K

L

M

N 21 2 e P 3: 0. R1 312~ 5 7b 17 13 21 35 51 1 5 2 25 1 9: 1 44:: T 53: 29: 21 3 V 55 113: W 21 256 57! x Y 1981 485 z 45: 39: J unknown(? notsequenced 4! 4: 2 2 2 2 2 2: 1 1 11 1 1 sum of seq' oomcaal mcaa' rel. oomcaal 53 53 5 5 5 55' 55: 55 55 565 6 5 6!5i ~5 7; 57; 57: 5 7: 53; 53: 29: 55; 35: 53: 53' 51* 32 521,25i 45: 39: 48 i52: 56: 441 57: 51: 57: T vS GS I S S Y YWSW R 0. OC) 5: C pos occupied'. 1 1 V 3: 3: 3 4 7 6. ~4 S 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320.

Table 6E: Analysis of V heavy chain subgroup 4 PCT/IEP96/03647 Framework 11 amino acid' M~ 0' N M LO W0 r- 0 5; 0 M J M t M Iq r ;t r qr LO V) in) in n LO~ A 8- 81:

B

D1 E 1 56 22: G 55: 55: 561: 1 i 5 7: H 21 24C 54 1.54 K 54! L 55 2

M

N 21: P 50::49 2: Q 56: R 9 1 S 3; 7. 1 52: T 11

V

W 56: x Y1: 15: 32: 23: z 57! 57: 57: unknown (P) not sequne sum or seq oomcaa' 5/ 57: 5/i 5/i 57i 57: 57t 57: 57. 57:i7 57:: 57: 57: 57:: 57. 57: 57: 57: 57: 56: 50: 4 9: 551 54* 55: 5 S5 56: 561 54156: 2 2: 54: 32: 57 57: 57: 24: 52: 57: mcaa' QP KG E I E YHS rel. oomcaa~ al al a o 0 60 0i 0 00 00: a) C CO O M: l 0 )M n O n: m: 6 a -r Ua pos occupied" 5: 2: 3: 2* 2* 2; 2 2* 3' 2 2! 6 1: 1: 1: 5 2 1: SUBSITUTJE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 6E: Analysis of V heavy chain subgroup 4 CDR 11 amino acid' 'L t'n L TO WE C' W' W (.00o W' TW aW r r7 r- r- nf- rQI

B

D 2: 1 E1 F 3:1 H K 153: 1: 1 L 1 5s 1 M 7::2 N 2 40: 53 2 P 1 54; 1

Q

R 2 3: 56: 2 S 49: 1 2: 56; 56 1 562 1:57; T 1::541 Ii I II 1 1 51: 1: 52: V 1153 2: x I. Y 11: 54' unknown() [not sequenced11111 1 sum of seqI oomrcaa, mcaa' rel. oomcaa" 57:. 57 40:; 54 56: 56: 56 53154: 56 57T 57::56 56: 56i 53 56: 57 5 1 48 5 7: 57: 57 i5 6: 50: 55 Y PS L S:R V T IS V DT S K 5 7: 5 7: 57: 5 2; 5 7: 51: pos occupied" 4 6: 2: 3 3 1 2: 3: 2 2 4 51 3: 4: 3: 5 1: 6: SUBSTITUTTE SHEET (RULE 26) WO 97/08320 PCT/EP96/03647 Table 6E: Analysis of V heavy chain subgroup 4 Framework [il amino acid' rrIco rl c) o&4< cOUQ 00 OC OC M A 55: 57: *57!

B

C 57: D 157: E1 F 54:1:.4 1

H

K 31 46G: 2:: L 1 1* 55S: 53: N 54: 3: 3:;1

P

Q 154 1 4 R 2.2 S t 1:57: 2 1 44:5 5: 1 2 T 1 4 53 V 2: 54: 1 551 x Y 5758SG unknown ,not sequenced sum of seq' i57 5 7 5 7: 57T 5 7 57 57 54: 54: 57: 55: 46i 53 571 57 44; 55 57: 57 54 :53 57: 57 551 57 5 7; 57: 57 571 55157 57: 5 7: 57.1 571 551 5 7: 561 571 oomcaa.1 incaa N 0 F S L K~ L A D~ T :V 71 rel. oomcaa' ao 000 0? 0: 0: 0 0 0 0 0 0O LnL: 0 ZD 0 0 C I y)l U' U' Cn rQ; C-0 LO: e D 0 0. 0 (D 0 CO 0 pos occupied' 21 2: 4~ 1 3. 8: 4: 7: 3: 3 3 3 2 1 3 1 2: 1: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6E: Analysis of V heavy chain subgroup 4 PCT/EP96/03647 CDR III amnino acid' CO U~ o0 C,3 .D Z A 5 6. 3! 3 21 4 2! 2; 41 2:1 1 11 11:12:1

B

C11 D 6: 55:;5::432;413::1 1 2:1 41: E 61 1 2:11 1 312:1: F 4- 1 1 2: 3; 2 2 1 131: G 2 5910:8:10:11: 47:;7: 6: 11: 12: 19 H 11112: I1 241 3:2:3 11 K 2 1 22:1 2 26735324:15S:3:3 1: M 1: 4 3 1: 2 N3: 21 11511 12; P 41:5311211 12:3:1 2:1 1 1 .1 R 5441225 53 2312 21 S 1148 8 12.,5::7:4:2:1 1:1 T I112:;1 344:3:3: 111 V 11422::5447::31112::1! W I1 21 22451 1 2 21 32: x Y 14 5316i4 23i:4 848 3 58 2 z 1 4 9::11 1623;:2729:34:3 1:14: 4i unknown not 3 3: 6 i71 8191 910: 11:11:11: 11 sum of seq' 57: 57: 561: 56: 56; 56: 56: 55; 54: 54: 51150: 491 48: 481 47: 46: 46: 4 6: 46G: see..

.0.

000* oomcaa mcaa rel. oomcaal pos occupied": 56542512 10810117: 911:16:23:27:29:34:31:14:31:41: ap a- a- a- a- a a 7tr.Lf:.4-: ID CJ C W N r r.

p..

2 4;1121 161 161 16i 16- 16' 16: 18: 18: 13: 151 13: 1098:5: 4t 4:: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6E: Analysis of V heavy chain subgroup 4 PCT/EP96/03647 Framework IV amino acid' 0 0 0 0 0 2 sum 1 A11

B

F

G. 401 H11 K 3: L 4; 19: M 9: N1 P 31 2: 2 Q 29 R 14: S 1 1 36:33 T 1 338: 34 12.. W 46: x Y 16: unknown not sequenced 10: 11: 16 17: 17: 20; 201 21: 21: 211 21: 22 332 113 210 176 135 674 282 278 540 43 204 281 334 250 986 532 488 267 455 466 4 426 *c, 0* bOgt~~ sum of seql oomcaa' mcaa, rel. oomcaa' pos occupied" 4 71 461: 41:: 40:: 40: 37; 37~ 36: 36; 36: 36:. 16: 46. 41 29: 40: 33 19 3 6: 3 4: 3G6 36t 33 Y W G 0 G T L V T V S 00 0~ 0~ M: C0: 4.~ 1 1 6~ 1 4: 1 3~ SUBSTITE SHEET (RULE 26) WO 97/09320 PCT/EP96/03647 Table GF: Analysis of V heavy chain subgroup 9

B

C1 D 2: E 881: 2: 4:9 3 92:

F.

1

H

96: K 94: 94: 77! L 1 91: 2! M 1:

N

P 1194: 3 2: 190:3 31 S 1921 94:

T

V 89 1 w x

Y

4..

unknown 1not sequenced 5: -51- 5: 5' 4: 4: 4 4 2 2: 2 2: 2: 2: 2 2: 2: 2: 1 sum of seq' oomcaa 1 921 92: 92:: 9 2: 93! 93:: 9 3i 9 3: 9 5* 95195 9 5 95:;95:: 95: 95:: 9 5: 95: 96: 96: 8890929189909292:89:93:9 1:94:94:94:94:92:94:95:77196 reomcaa~ pos occupied": 3 1~ 2: 4. 3 2 2: 4: 1~ 2: 1~ 4: U SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6F: Analysis of V heavy chain subgroup PCI'/EP96/03647 I CDRr amino acid' 7, C' c a N M Cl L W r A3 2:4: 8:

B

C 96 1 F 3: 6: 97: 2: G 192: 93:1 72: H14 4 K 89:1 L12 M11 N12: 4;14: 2: P1 Q 4: R 1 2 195: S 94: 1: 1901 84: 10::61: 2: 2: 15: 1 T 21 5: 7 516: 21: V 19 W93: 97: x Y 90 z 197: 97: unknown M) not sequenced 1 1I sum or seq' 0omcaa 1 mcaa' rel. oomcaa* pos occupiedr% W6 96: 961 96: 96; 96: 962 97: 97: 9797: 97: 97 97. 97; 97. 97: 97: 971 97: 94; 96:89: 92: 90! 93: 90184: 97: 7561: 97: 97;87: 9 3: 9 3 72:. 97: 931 SCK G S GY S F TS Y W I GWV R 0 0:C:on:. 2: 1: 5: 3 4: 3: 2: 8 i 5: 4. 5 1: 4: 3: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6F: Analysis of V heavy chain subgroup PCT/EP96O3 647 Framework 11 amino acid' C' -r 'V -Id Lo L-4 .0 nL A Vi G *96. K 1494 P 96 2 7 19:- G 97 16 15:6 96 1 7597997 no sequence sum or seq' oomcaa' inca a' rel. oomcaa pos occupied" 9 7 9 7:97:97 97i 97;97i 97:97: 97: 97:97: 97: 97197197197979797 97;: 92: 9 6' 97:' 94i 96 94i 97: 9 41 89 i9 5: 7 5: 92: 76: 931 9 7: 9 7: 691 931 9 6: 5 2: 1 2 3 2 3 5 61 5: 6: 4: 2: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6F: Analysis of V heavy chain subgroup PCT/EP96/03647 CDR 11 r. W M M Id, Ltn W N M Mn c0 U, U, 'n U, W (D C0 to0 W W. 01 r .N.N amino acid'

B

D 721 97: E 3: 2: 2 H 1 41 1 3: 8 91: L 1 4: 2: M3 N 2: 14i 2; P 951 9 81: R 781 3! 1 1: S: 2 9 519 5:!11 9 961: T 8 52: 1 96: 4: V 193: 2: 9 x Y 12: 92: unknownQ&) not sequenced sum of seq' oomcaa' inca a rel. oomcaal 97:97.97.97: 97. 97i 97i 97:197. 97: 9 7: 97: 97: 97: 97. 97 9 7: 9 7: 9 7 97T 7 7::857 892 9 5959591 91 94:8 193:96:88:95;88:97:93:96:91: o o ~0 a M: O !O OO0O!O iO V- r :B -e CO 0. LO a).

M. (M cn; CO wn (n (0 pos occupied": 6' 4 5 4: 3 3 31 4::1 3 21 1i 4: 4i .I 2 SUBS ITIUT11 SHEET (RULE 26) WO 97/08320 Table 6F: Analysis of V heavy chain subgroup PCT/EP96/03647 aminoacid' ~~ameor IIIj~ a r-~M V- L W CO a)C CD aaN A. 11 6 C 1 D 1 96: 3 H 3: 2: 9 K 9 11 L 96: 97: 2: N 7: 2! P1 1 9 31 S 87 2; 1 1: 90:91;9 T 2942 11 8 1: V 21 1 1 Y 94:: 94: 89: 94 unknown (P) not sequenced 2 4 4

S

4 sum of seq' oomcaa' 97:97:97:97;:97: 97;:9797971:9797!;97:97::97:971:97::9796:95 87 94!91 9496 93:95!90:91: 979 1:9619696::8893184:94:89::95: mcaa S T IA Y L:IQ W S IS L K A S 0 I1 A:M Y YC rl oc a, ,a ,a ,a a~a a, a, C,a pos occupied': 4: 3 5 2: 3: 3: 5 4 1: 5: 2 2: 2 41 2! 5: 2: 2: 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6F: Analysis of V heavy chain subgroup PCT/EP96/03647 I CDR Ill L r. Co On C) C J LL amino acid' ra M M 0) M2)<MC. L A 92:: 1 1: 1! 21 3: 31; 2: 1 .1 1. 4 2:

B

C 112: 1 D 33331:21:1:2: 211 2 37: E V1 11 21 11 1 1 F. 3: 32' 1 26: G 1: 9 11: 12: 12: 5 2: 4 3:10.2; 1: H 101 2: 11 1 3: 21211 1411 K 1 1 31 2: L 1 2: 3 1 1 2 5: 11* 1 M2: 1: 1: 1 V 1 1 1 N 1 2 112 1 2 5 1 3 1 1 1 Q 132 1 1 4 2 1 R 92; 7 9: 2' 2 2 1 2 S 1 1 3 26445 35 322 1 T 1 13211263 3 61 V 2 2 44 1 1 2 1 W 12 1 1 2 1 1 x Y 16369:8:7 2:12689 :9 0: z 1 1 2: 81 10: 16: 23* 30: 30: 31: 32: 30: 22: 7 2: unknown P) 11 not sequencedl 2 21525225 521 2:52:525225 55252:52:52: 5253:52: C 2 r n r A r A r~ r A A A A A AA: AC; .00.

0 a 0 SuII 0l SIN oomcaa'.

mcaa' rel. oomcaal 9 219 2:111 9:11 12: 12: 9 8: 10 16:23; 3030:31: 3230122126:37: AR L GG G GYY F D t-O g 0 0 0 O V' C) V r, N, D U)4 j a. CN pos occupied' 31 4! 131 161 14: 18: 161 15: 161 151 14:1111: 9: 8: 4: 6: 6: 4 I :1 1 SUBSTITUTE SHEET (RULE 26) WO 97108320 -PCTLEP96/03647 Table 6F: Analysis of V heavy chain subgroup Framework IV M jC Lfl LW fl CO 0 -cN amino acid' 0- 0 01 0 0: 0 c0: sumr

A

B

C

E1 F 2: G 4 1: 41::

H'

9 2 K 3: L 2: 251 M8

N

P 2:1 Q. 3.4: R ~1 S 2: i40: 39 T 1 408: 39: V 1 40: 41; W 43: x Y 13:: -2: unknown() ntsquenced -52! 54! 56156: 56! 56: 56: 56: 56: 56: 561 57 611 205 458 404 256 1065 44 588 650 549 303 64 414 612 351 1545 604 594 432 738 635 4 1678 sum of seq' oomc221 45:: 431 411: 41:: 41 131 43:: 41: 341 41 4 1: 411: 4114 11:41::411:40 40: 25: 40: 39; 41: 40N 39 t I mcaa, Y W G 0 G T LV TV S S 0: CD D rel. oomcaa" SO a;-o COC)C A5:C CO:~ K 0:00: M pos occupied' 10: ii' 4* 1 2 3 2 2 1: 2: 2: SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCT/EP96/03647 I Framework I q MN M (0 W M 5? z (N C 2 C' amino acid'

A

B

D

E

F.

G

H

K 68: L 5;68: 1:68

M

N

P 68! 67! R11 S521 1 68:1 661 T 168: 1 V 52 1 1 1166:1

Y

t unknown(? not sequenced 22:: 221 221 221221 2 2: 22: 221 66! 6: 6: 6: 6: 61 61 61 61 6: 6 sum of seq' 52: 52: 52: 52: 52; 52: 5 2: 5 2 68: 68: 68. 68: 68: 68: 68: 68: 68! 68: 68: 68 9

I

oomcaa' mcaa' rel. oomcaal 52152:: 52152151: 521 52 521 68! 67 68: 66: 681671 68: 681 68: 67: 66: 68: Q VQ LQOS GPIO L V K PS Q:T L S L 0 0 00;O 0; b 05 is~ 6 i a? C>Oa~r.

0: po. oc u ie I! V: 2: 2: 1. 1 2: 1: U P 2 3 1 SUBSITUTE SHEET (RULE 26) WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCT/EP96/03647 -7 ~CORII amino acid' c qQe'~ c'04ccN cr' ccN a4l cNm c. M. M' Cn c.0 A 1 67: 66::67:

B

D 68: 6

E

F 1 *6 2 H1 164: 2: 1 K 3:

L

M

N 1 2;66:

P

Q

S 1 69. 69: 1681 66: 1 3: T. V 1 4: 70. 6: 2 W 1 74: 7 4; x z unknown not sequenced 51 5. 5: 5: 5: 5: 51 5: 4: 4: sum of seq 2 69::69: 69: 69: 69: 69: 69: 69: 70: 70 74: 74: 74: 74: 74: 74: 74: 74: 74: 74:

S

S

*SS**S

oomcaa mcaa4 rel. oonicaa' 671 68 671 641 69; 691 68: 69: 70: 684 66: 66: 67: 66! 67: 74: 70: 7 4; 7 0 74: I CAI 6 0 SVS S A NW R 0 0 0D D r- '.c0 C50 ~0 0 0 f0 L pos occupied' 31 2: 3: 3 U1 1: 5: 6: 354 5 1 1 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table GG: Analysis of V heavy chain subgroup 6 PCT/EP96/03647 Framework 11 amino aCid' C' '0 04 Vfl 'I ;tUd O 1:.

B

C

E 7 4i 2 1 H1 K 11166: L 1 74: 74: P 73: R 73: 73: 72-:.

S 7 41 73: 1 72: T 1731

V

x S- 72: 72: z unknon) not sequenced sum of seq 7474741:74::74::7474::7474::74:74::74:74:74:74:74:74:7474774: oomcaa-' mcaa' 72: 74: 73: 73: 73: 741 741 74: 741 741 74. 731: 73i 721: 721: 721: 74:: 721: 66;: 73: Q S P S V -S.K W; rel.:VO 000000' ac I~ CS. a all 0 Ia- a pos occupied': 3: 11 2: 2: 2 11 1: 2: 2 21 31 3: 1 3 5 2 SUBSTITUTE SHEET (RULE 26) WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCTIEP96/03647 a m ino a c id' Wl W'S rat) 2 o w.0 T' 0r- L- n-.r A 73 1: G: C1 D 68:1 2 173: E 1 3: 7: 1 2: F 7 8 H11 1 65: 2:71:1 K 167: 1 L 15: *2 4:1 N 2 651 1 69: P 166: 1 R 13: 73: 52: 21 1 1 73: 1 66:1 2 1 73: T 4: 69: 1 71i 1 2! V 58. 72: 4: 2 1 x Y 601 unknown (M nLot sequencedI ^Fg, :l 7A 'A 7 7A:* 7A: 7A '7A 7A: 7A:. '7A 9 7A: 19 IA: IA: 11A: 4 @0 44 0@ 4 p tsp.

0@ ep 4

S

p p &too *00 oomcaa

I

60: 65 7 2: 7 3: 58: 73 7 21 67;: 66:: 7 31 6 5: 69 17 1: 69: 66; 73: 71 73: a mcaa' *Y N D rel. oomcaa- O C(4 Co Y AV S r- o a? Q On; On: r,O V KS R T TIN P D T S K 0 on 01; (O0in C On: m~ Tooc ld. 6: 5: 3 2: 7 2: 2: 5: 2~ 2: 4: 4: 3: 4: 21 4 2:; SUBSTITU TE SHEET (RULE 26) WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCT/EP96/03647 Framework III amino acid' r F o Q~ CO Q W:1 W WW1WM 1a A~ 74i

B

D 37 E 73: F 71 1 G1 H 2: 1

K

L 1 7 4: 72: .c 0 72 71 63 S 7717:1 73 7370 r 7 7 oomcaa) inca a' rel. oomcaas 7 2 71 747 4:7 1: 7 2631731731 737073:73:74:74;70:73:70173: Q F S L QL N SV T IP E D TAV YY C 07 0 1 0 03 o O 62 60 00000 a aa 0 n: tn:0 0 ~0 O O) 3311337222222113232 pos occupied' SUBSTIUTE SHEET (RULE 26) WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCT/EP96/03647 cnJ 0 Z:4 amino acid' A 69:: 1 1 3D 21: 41 31 2:

*B

D 19::43::7431 6111 62: E 10: 4: 2 1: 2 2: 1: 2:1 1 1 4 G 1 16 4: 15: 15- 11 8: 6 2 5: 1: 8 61 1 17: N 1312 1113 1 13 P 108:4: 253 51 1: 5:8 N 1* T: 1143.446131 2 1 3 V 0:4 3145195 4 9 11 R W9: 16 324 44 8 15 3* 5 5x' 7 7* S1 61 14 3 25' 33' 41' 47:3 4 7 5*~2 14 nosqu n e 1 2 2. 1 1 1: 1 1 1 1 1: 1; 1 12 12 1 2om aa 69 69 170 5 1 14: 23: 25: 33 41: 4 7: 53.: 54* 57: 56i 50: 2 8; 12: 6 a A D P. G potsocqupied 4 4: 1420191 17 16 16 13 13 1884 su f e' 1 1 7 7 2:7 2 2 7 2 2:-2J7o72 2 2 SUB T. UT SH E WO 97/08320 Table 6G: Analysis of V heavy chain subgroup 6 PCr/EP96/03647 L- Framework IV amino acid' 2 0 0 0 u A 2:

B

D1 F 2: 2: G 4 9: 50: H 2: K11 L 5 26: M 8

N

P 4: 6: o 40: R 2: 'S 4; 1 1 43:46 T 45S:4: 45: V 21V 246: 4 8 W 6 5:5 x Y 19! z 2: unknown() notsequenced 5: 8: 23: 24: 23; 24; 25 25 2825282 494 147 403 186 150 571 18 304 293 632 31 436 387 539 495 1271 640 647 398 518 585 13 580 1 Si sum of seq 7 oomcaa& 681 6 51 50: 49.: 50:: 49: 481: 48: 4 5: 4 8: 45: 4 7; 21: 65: 49: 40: 50; 45:26 46: 45; 48; 43; 46; V WGQ0G TL VT VS S mcaa4 rel. oomcaa I IS i a-, o) O:C 0: CN pos occupied, 9: 1 2 4: 1: 3: 7 W )0 0 eO oP 0 0 o 3 20 2 S SUBSTITUTE SHEET (RULE 26) WO 97/08320 WO 9708320PCT/EP96/03647 Appendix to Tables IA-C A. References of rearranged sequences References of rearranged human kaIjDQ sequences used for alignment 1 Alescio-Zonta, L. Et Baglioni, C. (1970) Eur.J.Biochem., 15, 450-463.

2 Andrews, D.W. Et Capra, J.D. (198 1) Biochemistry, 20. 5816-5822.

3 Andris, Ehrlich, Ostberg, L. Et Capra, J.D. (1992) J.lrnmunol., 149, 4053-4059.

4 Atkinson, Lamrpman, Furie, Naparstek, Schwartz, Stollar, B.D. at Furie, B. (1985) J.Clin.lnvest.. 75, 1138-1143.

Aucouturier, Bauwens, Khamlichi, Denoroy, L. Spinelli, Touchard, G., Preud'homme, J.-L Et Cogne, M. (1993) J.lmmunol., 1 50, 3561-3568.

6 Avila, Vazques, Danielsson, Fernandez De Cossio, M.E. Et Borrebaeck, CA.K.

(1993) Gene, 127, 273-274.

7 Barbas Iii, Crowe, Jr., Cababa. Jones, Zebedee, Murphy, B.R., Chanock, R.M. Et Burton, D.R. (1992) Proc.Natl.Acad.Sci.Usa, 89, 10164-10168.

8 Barbas, lii, et al. (1993) J-Mol-Biol., 230, 812-23.

9 Bentley, DL at Rabbitts, T.H. (1980) Nature, 288, 730-733.

Bentley, D.L at Rabbitts, T.H. (1983) Cell, 32. 181-189.

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2 Huber, Et Al. (1993) Eur.J.Immunol., 23, 2868.

3 Klobeck, Bornkammm, Cornbriato. Mocikat, Pohlenz, H.D. Et Zachau, H.G.

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Lorenz, Schabe, Thiebe, Stavnezer, J. at Zachau, H.G. (1988) Mol.lmmunol., 479.

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8 Pech, Jaenichen, Pohlenz, Neumnaier, Kiobeck, Et Zachau, H.G.

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9 Scott, Crimmins, Mccourt, DAW, Chung, Schable, Thiebe, Quenzel, Zachau, H.G. Et Nahm, M.H. (1991) J.Immunol., 147, 4007-4013.

Stavnezer. Kekish, Batter, Grenier. Balazs, Henderson, E. Et Zegers, BJ.M.

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11 Straubinger, Huber, Lorenz, Osterholzer, Pargent, Pech, Pohlenz, H.- Zimmer, Et Zacha L, H.G. (1988) J.Mol.Biol., 199. 23-34.

12 Straubinger, Thiebe, Huber, Osterholzer, E. Et Zachau, H.G. (1988) Biol.Chem.Hoppe-Seyer. 369, 601 -607.

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2 Siminovitch, Misener, Kwong, Song, Q.-L Et Chen, P.P. (1989) i.Clin.lnvest., 84, 1675-1,678.

3 Brockly, Alexandre, Chuchana, Huck, Lefranc, G. Ea Lefranc, (1989) 4 Daley, Pe ng, Misener, Liu, Chen, P.P. Et Siminovitch, K.A. (1992) Mol.lmmunol., 29, 1515-15S18.

Deftos. Soto-Gil, Quan, Olee, T. Et Chen, P.P. (1994) Scand. J. Immunol., 39, 6 Stiernholm, Kuzniar, B. at Berinstein, N.L (1994) J1 immunol., 152, 4969-4975.

7 Combriato, G. at Kiobeck,. H.G. (199 1) Eur.J.lmmunol., 21, 1513-1522.

*8 Anderson, Szajnert, Kaplan, Mccall, L. El Young, B.D. (1984) Nu c.Acids Res., 12, 6647-6661.

References of human germline heavy chain sequences 1 Adderson, Azmi, Wilson, Shackelford, P.G. at Carroll, W.L. (1993) J.lmmunol., 151, 800-809.

2 Andris, Brodeur, B.R. EtCapra, J.D. (1993) Mol.lmmunol., 30, 1601-1616.

242- SUBSTiTUTE SHEET (RULE 26) WO 97/08320l PCTIEP96/03647 3 Berman, Mellis, SJ., Pollock, Smith, Suh, Heinke, Kowal, Surti, U., Chess, Cantor, C.R Et Alt, F.W. (1988) Embo 7, 727-738.

4 Buluwela, L. Et Rabbitts, T.H. (1988) Eur-Jlmmunol., 18, 1843-1845.: Buluwela, L., Albertson, Sherring ton, Rabbitts, Spurr, N. Et Rabbitts, T.H. (1988) Embo 7, 2003 -20 Chen, Liu, Sinha, S. Et Carson, D.A. (1988) Arth.Rheum., 31, 1429-143 1.

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7 Cook, G.P. et al. (1994) Nature Genetics 7, 162-168.

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27 Tomlinson, Walter, CookEt Winter, G. (Unpublished) **eThe entire disclosure in the complete specification of our Australian Patent Application No. 68745/96 is by this cross-reference incorporated into the present specification.

B *.mSHE-u 6

Claims (9)

1. A modular vector comprising a cleavage site suitable for efficient ligation without modification with the modular nucleic acid sequences taken from the list of the human combinatorial antibody library (HgCAL) consensus genes: VKic (SEQ ID NO: 42), VK2 (SEQ ID NO: 44), VK3 (SEQ ID NO: 46), Vc4 (SEQ ID NO: 48), VX1 (SEQ ID NO: 50); V2 (SEQ ID NO: 52); VX3 (SEQ ID NO: 54); VH1A (SEQ ID NO: 56), VH1B (SEQ ID NO: 58), VH2 (SEQ ID NO: VH3 (SEQ ID NO: 62), VH4 (SEQ ID NO: 64), VH5 (SEQ ID NO: 66), VH6 (SEQ ID NO: 68), wherein the modules of said vector are flanked by restriction sites unique within said vector and essentially unique with respect to the restriction sites incorporated into said modular nucleic acid sequences, except for the restriction sites necessary for cloning said modular nucleic acid sequences into said vector.

2. A modular vector according to claim 1, wherein said modules are taken from the list comprising: origins of single-stranded replication, origins of double-stranded replication for high- and low copy number plasmids, promotor/operator, repressor or terminator elements, resistance genes, potential recombination sites, gene III for display on filamentous phages, signal sequences, purification and detection tags and sequences of additional moieties. S*

3. A modular vector according to claim 2, wherein said additional moieties are S taken from the list of: S a toxin, a cytokine, a reporter enzyme, a moiety being capable of binding a S'metal ion, a peptide, a tag suitable for detection and/or purification, or a homo- or hetero-association domain.

4. A modular vector according to any preceding claim, which is a cloning vector.

A modular vector according to claim 4, which is pMCS (SEQ ID NO: 264). o

6. A modular vector according to any one of claims 1 to 3, which is an expression vector. H:\Gabriela\Keep\Speci\13686-01.doc 26/05/2003 216

7. A modular vector according to claim 6, which is a vector suitable for expression and screening of libraries.

8. A modular vector according to any one of claims 1 to 3, which is taken from the list of: pCAL 4 (SEQ ID NO: 274), pCALO-1 (SEQ ID NO: 294), pCALO-2 (SEQ ID NO: 296), and pCALO-3 (SEQ ID NO. 299).

9. A modular vector according to claim 1, substantially as described herein with reference to the examples. Dated this 26 t h day of May 2003 MORPHOSYS GESELLSCHAFT FUR PROTEINOPTIMIERUNG mbH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *e H:\Gabriela\Keep\Speci\ 3686-01.doc 26/05/2003