Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57423—Specifically defined cancers of lung

Definitions

• the present invention relates to compositions and methods for detecting, managing or monitoring cancer.
• the invention also relates to antibodies specific for cancer markers, compositions and chips containing the same, as well as their uses for cancer detection, managing, monitoring, imaging or treatment, as well as for drug development.
• the invention is particularly suited for detecting, managing or monitoring lung cancer in human subjects. Background
• Lung cancer is the most common cause of death from cancer and each year 1.4 million new cases are diagnosed worldwide. More than two-thirds of lung cancers are diagnosed at a late stage, when clinical symptoms appear. The overall survival rate after diagnosis ranges from 14 % in the USA to 1.1 % in some regions of Asia and is currently very low due to the late diagnosis of the disease.
• Current diagnostics methods are based on imaging techniques and invasive procedures such as bronchoscopy or biopsy 2 .
• the few known plasma biomarkers for lung cancer such as carcinoembryionic antigen (CEA), cytokeratin- 19, squamous cell carcinoma antigen (SCC) and neuron-specific enolase (NSE) lack sufficient sensitivity and specificity 3 to be used as early diagnostics tools. Ongoing efforts using high throughput discovery technologies are still struggling to provide reliable and easily accessible lung cancer biomarkers to enter the clinic ' 5 .
• mAbs provide appropriately validated tools for the characterization and quantitative analysis of proteins but have primarily been targeted at a set of epitopes with low complexity ' .
• Global, antibody proteomics approaches aim to generate libraries of antibodies to cover most or all individual proteins and their immunogenic epitops in any complex proteome.
• Recombinant phage, bacterial and viral display represent approaches to global antibody generation but have had limited success in resulting of libraries capable to detect complex proteomes with sufficient quality affinity reagents and coverage 9 10 .
• the Human Protein Atlas and other large initiatives such as the NCI Clinical Proteomics Technology Initiative 12 are targeted at the generation of comprehensive libraries to the far more complex human proteome with both approaches using recombinant proteins as immunogens.
• the problem with recombinant proteins is that they do not represent the protein natural state, lack post-translational modifications and correct folding, therefore limiting the potential of the obtained libraries to profile natural proteomes.
• An object of the invention relates to particular antibodies, fragments or derivatives thereof, which bind cancer biomarkers, particularly lung cancer biomarkers. These antibodies, either alone or in combination, can be used to detect, manage or monitor cancer in a subject, particularly lung cancer.
• the invention also relates to kits or devices containing such antibodies, suitable for immunologic detection or reaction from any biological sample.
• the invention also relates to nucleic acids, vectors or cells, including hybridomas or recombinant cells, which produce antibodies of the invention.
• the invention is particularly suited to detect or monitor lung cancer in human subjects.
• FIG. 1 The major steps of the monoclonal antibody proteomics process. B
• 181 hybridomas were identified as candidates by showing a ratio higher than 1.5 between the two pools. These candidates were then tested with individual biotinylated depleted plasmas from a second small cohort (collection II) and the obtained candidates were further qualified on two larger cohorts (collection III and IV) of lung cancer samples and controls including non-related cancers and other lung diseases. These qualification steps led to the identification of 13 cloned hybridoma candidates suitable for further development of diagnostics tools.
• FIG. 1 Normalisation of plasma proteins.
• A The number of identified peptides for each protein identified in a shotgun proteomics experiment was plotted against the reported protein concentration in human plasma from the literature for depleted plasma ( ⁇ ) and three different conditions of normalization: (A T *) - loading of 1 mg protein at a flow lml/min
• B Complexity of protein fractions obtained from plasma shown by SDS-PAGE: total plasma (lane 1); depleted plasma using the affinity column Hu7 (lane 2) and Y12 (lane 4); the normalized plasma (lane 3) and glycosylated proteins enriched fraction fromY12 depleted plasma (lane 5).
• FIG. 3 Screening for lung cancer specific antibodies.
• A. Results from HT ELISA of the generated hybridomas with pooled plasma from lung cancer patients (y axis) are plotted against the results obtained with pooled plasma from the matched controls (collection I). Hybridomas considered as primary hits are shown as large circles; colour coding is according to the fusion performed to obtain the hybridomas.
• C. Statistical analysis of the screening results of the validated hits.
• FIG. 4 Receiver operating curves analysis for a single antibody (panel A) and the best panel determined from the data generated with 61 hybridoma supematants (panel B).
• the ROC for the panel was determined either on the testing data set only (50 % of all samples) shown as straight line, or for the entire dataset (including the training seta) shown as dotted line.
• the area under the curve which is a measure for the diagnostics capacity is shown for each curve.
• FIG. 5 High-throughput screening with nascent hybridomas.
• A. The same pool of depleted plasma from healthy donors was biotinylated and a HT-ELISA screening experiment with a panel of 88 mAb hybridoma supematants was performed 6 times on the same day and on 6 different days. The average and standard deviation from the results of the six experiments performed on the same day are plotted on the Y axis versus the average and standard deviations from the six experiments performed over 6 days (X-axis).
• B. The results of the screening experiment performed with two different purifications of the same antibody each spotted in four adjacent wells of the plate with 581 tracers (two tracers per plate) prepared from individual plasma samples.
• Plasma sample was split in two parts and two individual tracers were independently prepared (depletion and biotinylation) and tested with a panel of 88 mAb hybridoma supematants.
• Figure 6. Raw and transformed data from a HT-ELISA screening experiment. All screening data from an experiment performed with plasma collection III and 61 hybridoma supernatants (primary candidates) are plotted as the averaged Vmax (panel A) for each reaction. The results are presented per plate (in this case each plate is reacted with one plasma tracer); the positive controls are shown as red squares and the negative controls as blue squares.
• Panel B The results after normalization using positive and negative controls as described in Methods section "Screening data analysis".
• Figure 7 Algorithm used for building predictive model and determining best panel of biomarker able to discriminate lung cancer and control plasma.
• FIG. 8 Heavy chain V-region sequence of mABs.
• Bsi0033 SEQ ID NO: 77;
• Bsi0068 SEQ ID NO: 77;
• Bsi0076 SEQ ID NO: 82; Bsi0077: SEQ ID NO: 83; Bsi0080: SEQ ID NO: 84;
• Bsi0270 SEQ ID NO: 85; BsiO272: SEQ ID NO: 86; BsiO349: SEQ ID NO: 87; BsiO351 : SEQ ID NO: 88; BsiO352: SEQ ID NO: 89; BsiO358: SEQ ID NO: 90; BsiO359: SEQ ID NO: 85; BsiO272: SEQ ID NO: 86; BsiO349: SEQ ID NO: 87; BsiO351 : SEQ ID NO: 88; BsiO352: SEQ ID NO: 89; BsiO358: SEQ ID NO: 90; BsiO359: SEQ ID NO: 85; BsiO272: SEQ ID NO: 86; BsiO349: SEQ ID NO: 87; BsiO351 : SEQ ID NO: 88; BsiO352: SEQ ID NO: 89; Bsi
• FIG. 9 BSI0392 mAB. Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0392 in the plasma of control subjects and LC patients (A). SDS PAGE (reduced) followed by Western blotting (B).
• FIG. 10 BSI0352 mAB. Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0352 in the plasma of control subjects and LC patients.
• BSI0351 mAB Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0351 in the plasma of control subjects and LC patients.
• BSI0358 mAB Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0358 in the plasma of control subjects and LC patients .
• SDS PAGE reduced
• Western blotting of normal control and lung cancer plasma samples B. Immunohistological staining of paraffin embedded formaldehyde fixed tissue samples after antigen retrieval. Inserts are from HE stained tissue (2Ox primary objectives). Adenocarcinoma (upper) and squamous cell carcinoma (lower) panel. (C).
• BSI0359 mAB Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0359 in the plasma of control subjects and LC patients (A).
• B Figure 14.
• BSI0271 mAB Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0271 in the plasma of control subjects and LC patients (A).
• FIG. 15 BSI0033 mAB. Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0033 in the plasma of control subjects and LC patients (A). SDS PAGE (reduced) followed by Western blotting of a non reduced plasma sample. Note the haptoglobin specific ladder, which is due to polymerization of haptoglobin in the plasma (B).
• Figure 16 BSI0071 mAB. Relative individual (•) and descriptive statistics (box plots) of levels of cognate antigen recognized by mAB BSI0071 in the plasma of control subjects and LC patients (A).
• Figure 17. Histogram distribution of accuracy (AUC from ROC curves). Data from 408 patients (189 Ctrl & 219 LC) and 10 mAbs used in the analysis. LDA function with Leave- one-out cross validation. All possible combinations (1013) of the 10 mABs were tested.
• FIG. 19 ROC curve and formula classifiers for the panel of BSI0272, BSI0358 and BSI0392 mABs.
• Accuracy (AUC) is 0.89. Two thresholds; (i) with 0.876 sensitivity and 0.71 specificity and (ii) with 0.78 sensitivity and 0.88 specificity are marked.
• a challenge in the treatment of lung cancer is the lack of early, pre-symptomatic detection as lung cancer symptoms generally present at advanced stages.
• Further tests with plasma from patients with other inflammatory lung diseases and non-related cancers confirmed the lung cancer specificity of the monoclonal antibodies.
• the cognate antigen was identified for these antibodies, as well as binding peptides thereof.
• Fig. IA mAB proteomics
• Fig. IB complex antigen
• Hybridomas were obtained by PEG mediated fusion of spleen cells from the immunized mice with the Sp2/0-Ag-14 mouse myeloma cell line . Fused cells were seeded in microwells and their IgG production was estimated as the IgG concentration measured in the cell supernatants. IgG containing wells with at least 50 ng/ml IgG (27% of all fused cells) were then tested with biotinylated plasma tracers using direct ELISA in a series of screening experiments (Fig. IB) designed to identify antibodies capable of detecting lung cancer specific biomarkers.
• the screening experiments were based on direct ELISA using kinetic reading and the reproducibility of our assay (see Figure 5) allowed us to measure differences in the concentration of the antibody-antigen complex as low as 1.5. In the range of antigen concentrations below the saturation of the available antibody, the observed differences in the majority of the cases would be directly reflecting a difference in the biomarker concentration.
• the first three steps of the screening strategy aimed at identifying the antibodies with a good potential to discriminate cancer patients from control subjects. In the initial step, all IgG producing hybridomas from the two generated libraries were screened with pooled depleted and biotinylated plasma from twenty, apparently healthy control subjects and from twenty non-small cell lung-cancer patients (collection I) (Fig 3a).
• the plasma pooling was necessary to reduce the need for biological material (hybridoma supernatants and quantity of plasma) and to reduce the biological variability at this step 7 .
• the 184 nascent hybridoma supernatants selected to detect a differential ratio between the two pools higher than 1.5 were in their majority (87%) cancer specific.
• the next steps of screening were aimed at better qualifying the capacity of the selected antibodies to distinguish plasma from lung cancer patients from control subjects. This required screening experiments using tracers obtained from the individual plasmas.
• the plasma processing (depletion of highly abundant proteins and labelling with biotin) is highly reproducible (Figure 5C) and allowed us to compare signals obtained with the antibody candidates on hundreds of plasmas.
• Redundancy of the group of specific antibodies was assessed by V-region sequencing of the IgG, and the results confirmed independent clones for all of our candidates.
• the analysis of the functional redundancy showed several pairs of antibodies (e.g., BSI0070/BSI0072 and BSI0351/BSI0352) with similar reactivity and the results was confirmed by competitive ELISA experiments between these pairs that indicated the recognition of distinct epitopes on the same antigen by antibodies from each pair (data not shown).
• the cognate antigens for these 10 mAB have been identified, as well as binding peptide sequences thereof.
• Three mABs are specific for LRGl (Leucine rich glycoprotein 1), two mABs recognize Haptoglobin and Haptoglobin related protein (HRP), one mAB recognizes C9 (complement factor 9), two mABs recognize CHF (complement factor H), and two bind Alpha- 1-antichymo trypsin (ACT, Serpin A3).
• mABs also react with cancer cells in-situ, as detected by immunohistology, allowing their use for histopathological classification, immunological or immunohisto logical staining, and/or imaging of cancer.
• the invention thus provides novel antibodies which represent highly valuable reagents for cancer detection, management, diagnosis, monitoring, histopathological classification, staining, or imaging.
• the invention therefore relates to an antibody which binds a peptide having a sequence selected from SEQ ID NOs: 1 to 76 (see Table 4 below), or a fragment or derivative of such an antibody having the same antigen specificity.
• Binding peptides recognized by antibodies of the invention are disclosed below. These peptides have been tested in phage display ELISA. Bold peptide sequences also bind to the corresponding mABs in biotinylated form, immobilized to avidin coated 96 well plates, in direct ELISA experiments. Binding competition, displacement with human plasma and specific purified antigen was also confirmed for some peptides.
• the invention relates to an antibody which binds a polypeptide comprising a peptide sequence selected from SEQ ID NOs: 1 to 76, or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention relates to an antibody which binds a peptide selected from SEQ ID NO: 26-35 and 57-63 and which also binds a human LRGl (Leucine Rich Alpha -2 Glycoprotein 1) protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention particularly relates to an antibody which binds a peptide selected from SEQ ID NO: 26-35 and 57-63 and wherein said binding is at least partially displaced by a human LRGl protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• Lung cancer specific antibodies binding to other peptide sequences have also been isolated.
• Corresponding antibodies and peptides are listed in table 6 (SEQ ID NO: 93-135).
• the invention also relates to an antibody which binds a peptide having a sequence selected from SEQ ID NOs: 93-135 (see Table 6), or a fragment or derivative of such an antibody having the same antigen specificity.
• Another particular preferred embodiment of the invention relates to an antibody which binds a peptide selected from SEQ ID NOs: 14-25 and which also binds a human C9
• (Complement component 9) protein or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention particularly relates to an antibody which binds a peptide selected from SEQ ID NOs: 14-25 and wherein said binding is at least partially displaced by a human C9 protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• a particularly preferred antibody of the invention binds a peptide selected from SEQ ID NOs: 14-22 and 24 and binds a human C9 protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• Another particular preferred embodiment of the invention relates to an antibody which binds a peptide selected from SEQ ID NOs: 36-38 and which also binds a human haptoglobin or haptoglobin-related protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention particularly relates to an antibody which binds a peptide selected from SEQ ID NOs: 36-38 and wherein said binding is at least partially displaced by a human haptoglobin or haptoglobin-related protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• Another particular preferred embodiment of the invention relates to an antibody which binds a peptide selected from SEQ ID NOs: 43-56 and 64-76 and which also binds a human CFH (Complement factor H, formerly known as Beta IH globulin) protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention particularly relates to an antibody which binds a peptide selected from SEQ ID NOs: 43-56 and 64-76 and wherein said binding is at least partially displaced by a human CFH protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• a particularly preferred antibody of the invention binds a peptide selected from SEQ ID NOs: 64-71, 73 and 75-76 and also binds a human CFH protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• Another particular preferred embodiment of the invention relates to an antibody which binds a peptide selected from SEQ ID NOs: 1-13 and 39-42 and which also binds a human Alpha- 1-antichymo trypsin protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• the invention particularly relates to an antibody which binds a peptide selected from SEQ ID NOs: 1-13 and 39-42 and wherein said binding is at least partially displaced by a human Alpha- 1-antichymo trypsin protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• a particularly preferred antibody of the invention binds a peptide selected from SEQ ID NOs: 1-5 and 7-12 and also binds a human Alpha- 1 -antichymo trypsin protein, or a fragment or derivative of such an antibody having the same antigen specificity.
• antibodies of this invention comprise all or part of a heavy chain variable region sequence selected from SEQ ID NOs: 77-92 ( Figure 8).
• the polypeptide may be or may comprise a complete variable region of an antibody, or may comprise only a part thereof, such part preferably comprising at least 5 consecutive amino acid residues, more preferably at least 6, 7 or 10 consecutive amino acid residues.
• a preferred polypeptide of the invention is a polypeptide comprising at least a CDR or FR domain of any one of SEQ ID NOs: 77-92.
• the FR domains are represented in blue (or grey) on Figure 8, while the CDR domains are represented in white.
• Variant sequences wherein from 1 to 10 amino acid residues have been replaced, deleted or inserted are also included in the present invention, as long as the modification does not substantially alter antigen binding capacity or specificity of the sequence or domain.
• antibodies of the invention typically contain 1, 2 or 3 amino acid modifications, typically consisting of replacement with amino acid of same nature.
• Illustrative and preferred examples of antibodies of the invention include monoclonal antibodies BsiO358, BsiO359, BsiO272, BsiO392, Bsi0080, BsiO352, Bsi0077, BsiO349, Bsi0270, BsiO271, Bsi0072, Bsi0076, Bsi0068, Bsi0033, Bsi0071, Bsi351, or Bsi0070, or a fragment or derivative thereof having the same antigen specificity.
• These antibodies comprise a variable region sequence as disclosed in SEQ ID NOs: 77-92, respectively.
• a further object of the invention is a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 76.
• a further object of the invention is an isolated nucleic acid encoding an antibody of the invention, or the fragment or derivative of said antibody.
• the nucleic acid typically encodes at least a portion of a variable region of the antibody, e.g., a portion of the heavy or light chain including a variable domain, such as a CDR or FR domain.
• the nucleic acid may be DNA or RNA, including cDNA, gDNA, recombinant DNA, synthetic or semi-synthetic DNA, which may be single- or double-stranded.
• a particular object of the invention is a nucleic acid encoding a polypeptide comprising at least a CDR or FR domain of any one of SEQ ID NOs: 77-92.
• the nucleic acid may be fused to other regions (e.g., constant regions, hinge regions, etc) to create synthetic antibodies, humanized antibodies, chimeric antibodies, etc., according to techniques well known per se in the art. They may also be used to produce single chain antibodies.
• the invention relates to antibodies (or fragments or derivatives thereof) which "bind" peptides or polypeptides.
• binding should be specific or selective, meaning that the binding to the reference peptide or polypeptide can be discriminated from (e.g., occurs with higher affinity or avidity than) possible non specific binding to other antigens.
• Preferred antibodies do not bind, under selective condition, to any other unrelated human blood protein but the reference protein. Binding of an antibody to the above reference peptide can be tested as disclosed in the examples. Binding to a peptide can be verified with either the isolated peptide (e.g., immobilized on a support) or with the peptide included in a larger polypeptide sequence.
• the peptide is in isolated form and immobilized on a support (e.g., a plate) and the candidate antibody is incubated with the immobilized peptide. Binding may then be revealed using known techniques. Binding to a protein may be tested by incubating any sample containing the protein in solution, and verifying the formation of an immune complex.
• the term "binding" to a peptide indicates the antibody can bind the corresponding peptide in biotinylated form, immobilized to avidin coated well plates in direct ELISA experiments.
• the antibody may be a polyclonal or a monoclonal antibody, most preferably a monoclonal. It may be of various classes (e.g., IgG, IgE, IgM, etc.). The antibody may be of various animal origin, or human or synthetic or recombinant. Furthermore, the term antibody also includes fragments and derivatives thereof, in particular fragments and derivatives of said monoclonal or polyclonal antibodies having substantially the same antigenic specificity. Antibody fragments include e.g., Fab, Fab '2, CDRs, etc). Derivatives include humanized antibodies, human antibodies, chimeric antibodies, poly- functional antibodies, Single Chain antibodies (ScFv), etc.
• ScFv Single Chain antibodies
• polyclonal antibodies may be produced according to conventional methods, including immunization of an animal and collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with appropriate cell lines).
• Methods of producing polyclonal antibodies from various species, including mice, rodents, primates, horses, pigs, rabbits, poultry, etc. may be found, for instance, in Vaitukaitis et al.
• the antigen is combined with an adjuvant (e.g., Freud's adjuvant) and administered to an animal, typically by sub-cutaneous injection. Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separared.
• an adjuvant e.g., Freud's adjuvant
• Recombinant antibodies of the invention may be produced by methods known per se in the art, for example by recombination in a host cell, transformed with one or more vectors enabling the expression and/or secretion of the nucleotide sequences encoding the heavy chain or the light chain of the antibody.
• the vector generally contains a promoter, translation initiation and termination signals, and suitable transcriptional regulatory regions. It is stably maintained in the host cell and may optionally possess specific signals for secretion of the translated protein. These different components are selected and optimized by one of skill in the art according to the host cell used.
• Another object of the invention is an expression vector, for example a viral or plasmid vector, comprising a nucleic acid of the invention.
• the vector may replicate autonomously in the chosen host cell, or it may be an integrative vector.
• an expression vector comprising a nucleic acid coding for the light chain of the antibody.
• Another object of the invention is an expression vector comprising a nucleic acid coding for the heavy chain of an antibody of the invention.
• Such vectors are prepared by methods known per se in the art, and the resulting clones may be introduced into a suitable host cell by standard methods, such as lipofection, electroporation, use of polycationic agents, heat shock, or chemical methods.
• the host cell may be selected from among prokaryotic or eukaryotic systems, for example bacterial cells but also yeast cells or animal cells, in particular mammalian cells. Insect cells or plant cells may also be used.
• Another object of the invention is a hybridoma cell producing an antibody of the invention.
• the invention in another aspect, relates to a method for producing an antibody of the invention, said method comprising the following steps : a) culturing in a suitable culture medium a host cell expressing a heavy chain and/or a light chain such as defined herein; and b) recovering said antibodies so produced from the culture medium or from said cultured cells.
• a suitable culture medium a host cell expressing a heavy chain and/or a light chain such as defined herein
• recovering said antibodies so produced from the culture medium or from said cultured cells a method for producing an antibody of the invention, said method comprising the following steps : a) culturing in a suitable culture medium a host cell expressing a heavy chain and/or a light chain such as defined herein; and b) recovering said antibodies so produced from the culture medium or from said cultured cells.
• H chain heavy chain
• L chain light chain
• a double-recombinant vector may be prepared in which the sequence encoding each of the H and L chains is under the control of a
• a particular example of a production method is production in an insect cell, as described for example in international patent application WO 96/07740.
• an expression cassette is used comprising a sequence coding for the variable region of the monoclonal antibody light chain, or a sequence coding for the variable region of the monoclonal antibody heavy chain, said sequence is placed under transcriptional control of a suitable promoter, for example a baculovirus promoter.
• a production method is the use of a viral or plasmid expression vector for expressing the monoclonal antibody in a mammalian cell.
• Preferred mammalian cells for expressing the monoclonal antibody are the rat YB2/0 line, the hamster CHO line, in particular the lines CHO dhfr- and CHO Led 3, PER.C6TM (Crucell), 293, K562, NSO, SP2/0, BHK or COS, C0S7.
• a further production method is the expression of the recombinant antibody in transgenic organisms, for example in plants (Ayala M, Gavilondo J, Rodriguez M, Fuentes A, Enriquez G, Perez L, Cremata J, Pujol M. Production of plantibodies in Nicotiana plants. Methods MoI. Biol. 2009; 483: 103-34) or else in the milk of transgenic animals such as rabbit, goat or pig (Pollock, D.P., J.P. Kutzko, E. Birck- Wilson, J.L. Williams, Y. Echelard and H. M. Meade. (1999) Transgenic milk as a method for the production of recombinant antibodies. Journal of Immunological Methods. 231: 147-157).
• the antibodies of the invention may be coupled to heterologous moieties, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers.
• Labels include radiolabels, enzymes, fluorescent labels, magnetic particles and the like.
• Toxins include diphteria toxins, botulinum toxin, etc.
• Drugs or therapeutic agents include lymphokines, antibiotics, antisense RNA or antisense nucleic acid, modified or not, growth factors, etc. Methods of using such heterologous moieties are illustrated, for instance, in US4,277,149 and US3,996,345.
• the antibodies of this invention have various applications, including, diagnostic, purification, detection, therapeutic, prophylactic, etc.
• these antibodies In vitro, they can be used as screening agents or to purify the antigen from various samples, including various biological samples (e.g., blood samples). As demonstrated in the examples, these antibodies have the remarkable property of binding to antigens which are differentially expressed between cancerous and control human subjects.
• the invention thus relates to a diagnostic composition
• a diagnostic composition comprising an antibody (or a fragment or derivative thereof) as defined above.
• the composition may comprise any excipient or solid support.
• the invention also relates to a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody as defined above and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• the method preferably comprises contacting said sample from said subject with at least two antibodies as defined above, preferably at least 3, in combination.
• the invention discloses particular antibody combinations which allow specific and sensitive determination of lung cancer in human subjects. These combinations include, without limitation:
• a preferred type of combinations comprises BsiO392 in combination with at least one or two additional antibodies.
• Another preferred type of combinations comprises Bsi0071 in combination with at least one or two additional antibodies.
• Another preferred type of combinations comprises BsiO272 in combination with at least one or two additional antibodies.
• Another preferred type of combinations comprises BsiO358 in combination with at least one or two additional antibodies.
• Another preferred type of combinations comprises Bsi0077 in combination with at least one or two additional antibodies.
• Another preferred type of combinations comprises at least one anti-C9 antibody in combination with at least one or two additional antibodies.
• the term "combination” indicates the sample should be tested for antigen binding to all antibodies of the combination, either simultaneously or separately (e.g., sequentially). Preferably, the antibodies are tested simultaneously (e.g., on the same device).
• a further object of this invention resides in a device comprising at least one antibody as defined above immobilized on a support.
• the support may be, e.g., a membrane, a slide, a microarray, a chip or a microbead. Immobilization can be made through techniques known per se in the art (using linkers, cross linking reagents, passive adsorption, etc.).
• a further object of the invention resides in a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody that binds a Leucine-Rich alpha-2 glycoprotein (LRGl) and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• a sample from said subject preferably a blood sample
• at least one antibody that binds a Leucine-Rich alpha-2 glycoprotein (LRGl) determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• a further object of the invention is a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody that binds a haptoglobin (HP) or HRP protein, and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• a sample from said subject preferably a blood sample
• a further object of the invention is a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody that binds a C9 protein and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• a further object of the invention is a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody that binds a CFH protein and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• a further object of the invention is a method for detecting lung cancer in a subject, the method comprising contacting a sample from said subject, preferably a blood sample, with at least one antibody that binds an Alpha- 1 -anti chymotrypsin (ACT, SERPINA3) protein and determining the presence of an antigen bound to said at least one antibody, said presence being indicative of lung cancer.
• Preferred methods comprise contacting said sample from said subject with at least two antibodies which bind a distinct protein selected from Leucine -Rich alpha-2 glycoprotein, haptoglobin, haptoglobin related protein, C9, CFH or Alpha- 1-antichymo trypsin.
• Further preferred methods comprise contacting said sample from said subject with at least three distinct antibodies which bind a distinct protein selected from Leucine-Rich alpha-2 glycoprotein, haptoglobin, C9, CFH or Alpha 1 Antichymotrypsin.
• antibody as used therein also includes fragments and derivatives thereof as defined above.
• a further object of the invention is a device comprising at least one antibody that binds a protein selected from Leucine-Rich alpha-2 glycoprotein, haptoglobin, haptoglobin related protein, C9, CFH or Alpha- 1 -antichymotrypsin, immobilized on a support.
• a further object of the invention is a device comprising at least two antibodies that each bind a distinct protein selected from Leucine-Rich alpha-2 glycoprotein, haptoglobin, C9, CFH or Alpha-1-antichymotrypsin, immobilized on a support.
• the support may be a membrane, a slide, a microarray, a chip or a microbead based detection system.
• a further object of the invention is a kit comprising a device as defined above and a reagent to perform or detect (or quantify) an immune reaction, particularly an antibody-antigen complex.
• Reagents include labels, buffers, substrates, etc.
• the kits typically comprise containers for the different reagents and products, and may further comprise a support or other device suitable to perform the assay.
• the antibodies can be used individually or in combination to measure the level of the cognate antigen (analyte) in biofiuids including serum, plasma, urine, cerebrospinal fluid, bronchoalveolar lavage (BAL) fluid, sputum, tear, sweat, amniotic fluid and inflammatory exudate, using any number of detection technologies or platforms such as, without limitation Capture assay, Sandwich assay, Competition assay, Radio-immuno assays, Enzyme labels with substrates that generate colored, fluorescent, chemiluminescent, or electrochemically-active products, Fluorescence, fluorescent polarization, Chemiluminescence, Optical and colorimetric, Electrochemiluminescence, Time -resolved fluorescence, Surface plasmon resonance, Evanescent wave, Multiwell plate (ELISA), Individual assay, Multiplex assay, Latex bead - multiplex assay, Microarray (Laminar surface) - multiplex assay, Glass, Ceramic (like Randox), Plate based
• the antibody is immobilized directly to the support or captured by an affinity reagent such as an anti-mouse IgG antibody coated onto the support.
• the immobilized antibody is then incubated with any of the above mentioned body fluids in which the proteins have been labeled with a detection molecule such as biotin, with or without pre -treatment to remove abundant proteins.
• a detection molecule such as biotin
• the labeled protein which is bound by the antibody is detected by the addition of an appropriate detection reagent which binds to the label such as avidin or streptavidin which has been modified to be compatible with one of the detection technologies described in the section "detection technology.”
• the first antibody is immobilized directly to the support or captured by an affinity reagent such as an anti-mouse IgG antibody coated onto the support.
• the immobilized antibody is then incubated with any of the above mentioned body fluids, with or without pre-treatment to remove abundant proteins.
• the antibody/antigen complex is then incubated with a second antibody, made against the same protein, which has been labeled with a detection molecule such as biotin.
• the bound antibody is detected by the addition of an appropriate detection reagent which binds to the label such as avidin or streptavidin which has been modified to be compatible with one of the detection technologies described in the section "detection technology.”
• the resulting signal provides a quantitative measure of the amount of protein bound by the antibody
• the antibody is immobilized directly to the support or captured by an affinity reagent such as an anti-mouse IgG antibody coated onto the support.
• the immobilized antibody is then incubated with any of the above mentioned body fluids.
• the immobilized antibody/antigen complex is then incubated with a labeled tracer consisting of either (1) any of the above mentioned body fluids in which the proteins have been labeled with a detection molecule such as biotin, with or without pre -treatment to remove abundant proteins, or (2) a purified or recombinant protein recognized (bound) by the monoclonal antibody, or (3) a peptide which is recognized (bound) by the monoclonal antibody.
• the labeled protein or peptide which is bound by the antibody is detected by the addition of an appropriate detection reagent which binds to the label such as avidin or streptavidin which has been modified to be compatible with one of the detection technologies described in the section "detection technology.”
• Preferred Detection Technologies include:
• Enzyme labels with substrates that generate colored, fluorescent, chemiluminescent, or electrochemically-active products are enzyme labels with substrates that generate colored, fluorescent, chemiluminescent, or electrochemically-active products .
• the detection reagent (for example steptavidin or avidin, which binds to biotin) is coupled to an enzyme such as horseradish peroxidase which is capable of catalyzing o an appropriate colorimetric substrate of which the product demonstrates maximal absorbance at a given wavelength allowing the quantitative measurement of the labeled protein by measuring the optical density of the final product in the well at or near the wavelength of maximal absorbance.
• an enzyme such as horseradish peroxidase which is capable of catalyzing o an appropriate colorimetric substrate of which the product demonstrates maximal absorbance at a given wavelength allowing the quantitative measurement of the labeled protein by measuring the optical density of the final product in the well at or near the wavelength of maximal absorbance.
• o a chemiluminescent substrate to a sensitized reagent which upon oxidation emits light, providing the quantitative measurement of the labeled protein.
• a chemiluminescent substrate to a sensitized reagent which upon the application of an electrical current emits light,
• the detection reagent for example steptavidin or avidin, which binds to biotin
• the detection reagent is coupled to a fluorescent tag.
• Preferred platform Technologies include:
• o Single test one antibody is immobilized per well either directly or indirectly using a capture reagent such as goat anti-mouse antibody.
• o Multiplex 2 or more antibodies are immobilized in a single well by deposition in a pattern
• Two or more antibodies are immobilized onto a latex bead between x and y microns
• Two or more antibodies are spotted onto an activated laminar surface with a spot diameter between 100 ⁇ m - 5 mm (arrays), 2 ⁇ m - 100 ⁇ m (microarrays), 10 nm-2 ⁇ m (nano-arrays)
• the surface can be composed of glass, plastic, ceramic, carbon nanotube lattice etc.
• the method can be performed at any stage of the disease, such as early or late stage, to confirm or reject a prior diagnosis, select patients for surgery, classify cancer type or severity, or monitor patients. The test may also be conducted before disease symptoms, as a first line detection.
• the test will avoid futile thoracotomies and unnecessary and expensive imaging technologies that are not specific enough and expose the patients to potentially harmful irradiation, and missed cures as observed patients could receive the test repeatedly.
• Plasma specimens were in part purchased from Proteogenex (Culver, CA) and Asterand (Royston, UK). Other samples (see Table 1) were collected at the Department of Pulmonology of the University of Debrecen in Hungary from informed and consented patients and matched (age, sex and smoking habit) apparently healthy individuals by a clinical protocol RKEB/IKEB:2422-2005 approved by the regional ethics committee and the IRB of the clinic.
• a plasma sample (250 uL) was thawed and diluted by addition of 750 uL of buffer A (25 mM Tris, 0. 5M NaCl, ImM MnC12, ImM CaC12 and 0.05% sodium azide, pH 7.4).
• buffer A 25 mM Tris, 0. 5M NaCl, ImM MnC12, ImM CaC12 and 0.05% sodium azide, pH 7.4
• the diluted plasma was loaded onto the Seppro 12 column at a flow rate of 0.5mL/min for 30 min.; the flow rate was then increased to 2 mL/min for the remainder of the run.
• the unbound proteins (depleted fraction) were washed off with binding buffer and the depleted fraction was collected into a 15 mL centrifugal filter Amicon with a cut-off at 5kDa.
• the depleted plasma was concentrated by centrifugation at 3,500 x g.
• the bound proteins were eluted from the column with stripping buffer (100 mM glycine, pH 2.5) and collected into a separate tube.
• the column packing material was neutralized with 100 mM Tris-HCl, pH 8.0 for 10 min and re-equilibrated with binding buffer, before performing protein depletion from the next plasma sample.
• a total of 27 depletion runs for the normal plasma and 20 runs for the lung cancer plasma were performed.
• the concentrated and depleted fractions from each group were pooled before further processing. - depletion of high abundant proteins from individual plasma samples for tracers preparation
• the depletion of the seven most abundant proteins was performed using commercially available Human-7 Multiple Affinity Removal System columns (10X100mm) from Agilent Technologies (Santa Clara, CA). The process was automated using a chromatography system AKTATMpurifier 10 - collector F950" from GE Healthcare (Chalfont St. Giles, UK) connected to an autosampler A-900. The individual plasmas (70 uL) were diluted four times with the vendor provided equilibration buffer (buffer A) and filtered using 0.22 ⁇ m spin filters at 16000 g for 1 min. The filtered plasmas were loaded on the column at a flow rate of 0.
• MARS Hu-7 columns were used according to the vendor specifications for maximum of 200 runs and their performance was followed at regular intervals (each 40 runs) for protein leaking (albumin, IgG, IgA and fibrinogen) by an ELISA using commercially available mAbs.
• Glycoprotein enrichment was performed using a multi-lectin affinity chromatography (M- LAC) column.
• M- LAC multi-lectin affinity chromatography
• the lectin column contains a mixture of 3 lectins: Con A, WGA and JAC from Vectors Laboratories (Burlingame, CA) and was prepared in-house using Aldehyde POROS- 20 AL® (20 ⁇ m beads) beads from Applied Biosystems, (Foster City, CA) as previously reported 15 .
• One mL of the pooled depleted plasma sample was loaded on a 7.8 mL M-LAC column (10 x 100) at a flow rate of 0.5 mL/min for 20 min.
• the flow rate was increased to 4 mL/min, and the unbound proteins (flow through fraction) were washed-off with M-LAC binding buffer (25 mM Tris, 0. 5OM NaCl, ImM MnCl 2 , ImM CaCl 2 and 0.05% sodium azide, pH 7.4) for 10 min.
• M-LAC binding buffer 25 mM Tris, 0. 5OM NaCl, ImM MnCl 2 , ImM CaCl 2 and 0.05% sodium azide, pH 7.4
• the flow through fraction was collected and stored at -75°C.
• the proteins which bound to the M-LAC were eluted with 100 mM acetic acid, pH 3.8 at 4 mL/min for 10 min.
• the eluted glycoproteins were collected directly into a 15 mL Amicon filter device and concentrated as described above.
• the sample was buffer exchanged into IX PBS by addition of 14 mL of buffer to the filter device; the volume was reduced down to ⁇ 1.0 mL by centrifugation; this step was repeated twice.
• the M-LAC column was neutralized with 0.5 M Tris, pH 7.5, IM NaCl, 0.05 % sodium azide and equilibrated prior to the next run.
• the bound glycoproteins from 5 runs were pooled, aliquoted and stored at -75°C. The total glycoprotein yield was approximately 5.0 mg for the lung cancer and 3.0 mg for the matched control samples.
• Protein normalization of the depleted plasma and the glycosylated protein fraction was performed using an in-house immunoaffinity column prepared as follows: rabbit polyclonal antibodies raised against normal human serum from Sigma (Saint Louis, MO) were covalently linked to HiTrap Protein G HP column (4.6 x 100) from GE Healthcare (Chalfont St. Giles, UK) using 15 mM dimethyl pimelimidate and 15 mM dimethyl suberimidate as previously described .
• HiTrap Protein G HP column 4.6 x 100
• GE Healthcare Chalfont St. Giles, UK
• Ix PBS buffer pH 7.0
• the flow through represented the normalized protein fraction, while the bound proteins were eluted with stripping buffer B from Agilent Technologies (Santa Clara, CA).
• the column was equilibrated with Ix PBS.
• the pooled or individual depleted plasma was labeled with a bifunctional NHS-biotin having a long alkyl chain as a spacer EZ-Link Sulfo-NHS-LC-Biotin from Pierce (Rockford, IL). Labeling was performed in PBS buffer (pH 7.0) at a 100 time molar excess of biotin assuming an average protein mass of 68 kDa for 30 min. at room temperature. The non-reacted protein was removed using a 5 ml HiTrap desalting column HP from GE Healthcare (Chalfont St. Giles, UK) with PBS (pH 7.0) at a flow rate of 1 ml/min. Immunisation of BALB/c mice
• mice Two groups of four female Balb/c mice of at least 8 weeks of age from Charles River Laboratories (Evry, France) were injected subcutaneously in the rear footpads and at the base of the tail with the two complex antigen protein mixtures. Each mice received 10 ⁇ g protein of glycoprotein enriched depleted plasma (group A) and normalized glycoprotein enriched depleted plasma (group B) on days 1, 15 and 29. Complete Freund's adjuvant (Sigma, Saint Louis, MO) was used in all cases. Blood was taken from each mouse by retro- orbital bleed using a Pasteur pipette on days 19 and 33 to monitor antibody production by ELISA.
• reaction development was carried out by adding 20 ⁇ l freshly prepared substrate solution to each well (o-phenylenediamine at 0.4 mg/ml in 0.05 M phosphate/citrate buffer pH 5.0). The kinetics of the reaction development at 37 0 C was followed at 450 nm by recording the absorbance multiple times.
• Liquid handling was performed using Multidrop Combi from Thermo (Waltham, MA), Multimek with 96 pin head from Beckman Coulter (Fullerton, CA) and STAR from Hamilton (Reno, NV). Plate washing was performed using ELX405 from BioTek (Winooski, VT). Absorbance was measured with a microplate reader SpectraMax from Molecular Device (MDS, Toronto, Canada).
• Vmax of the chromogenic reactions were calculated from the linear part of the kinetic readings using the software provided with the plate reader SoftMax Pro from Molecular Device (MDS, Toronto, Canada). Each plate had eight positive and negative controls used to calculate Z' factor, a metrics used to quantify the quality of the screening experiment with respect to reproducibility and data scatter . Plates with a Z' factor below 0.5 (usually less than 10% in a screening campaign) were repeated. The positive (PC) and negative controls (NC) were used to normalize the data across plates and according to the following formula:
• VmaxN sam pie (Vmax samp i e -Vmax NC )/ (Vmax PC -Vmax NC ).
• Aberrant data (outliers) for each group of replicates were removed using automated procedure based on the mean and standard deviation values of the multiple measurements.
• CF is a normalized measure of dispersion of the probability distribution and it is defined as the ratio of the standard deviation ⁇ to the mean ⁇ as follows:
• T max (i) ⁇ (i) + 1.3* ⁇ (i)
• the dataset was partitioned randomly into two parts (Figure 7), a training dataset and a testing dataset with a respective size of 50%(75%) and 50%(25%) of the available cancer and control samples from collection III.
• the classifiers were built and tested on the training set and the best one was finally applied on the testing data set to determine the diagnostic accuracy of the classifier.
• Selection of hybridomas for building the classifier was carried out by ranking the hybridomas according the p-values obtained for each one of them in comparing the results between the two groups (control vs lung cancer) using the Mann- Whitney test.
• LDA Linear Discriminant Analysis
• AUC area under the receiver operating curve
• the R statistical software (available from www.cran.r-project.org) was used for the statistical analysis and for the implementation of the algorithm.
• the Ph.D-12TM Phage Display Peptide Library Kit was obtained from New England Biolabs (Beverly, MA). The phages display random peptide 12-mers are fused to a minor coat protein (pill) of M13 phage. The library consists of approximately 2.7x10 sequences. The experiments were carried out as described in the Instruction Manual of the Phage Display Kit with minor modifications. Phages were propagated using the supplied E. coli (ER2738) host strain. 96-well microtiter plates (Maxisorp Cat. no. 442404, Nunc, Napperville, IL) were coated with the mapped mAbs (10 ⁇ g antibody in 100 ⁇ l 0.1 M NaHCO 3 pH-8.6/well).
• 4xlO 10 phages were added in TBS-Tween to the wells and incubated for 1 hour at room temperature. Bound phages were eluted by lowering the pH (0.2 M Glycine -HCl pH 2.2). Recovered phages were amplified and their titer determined. The selection was repeated twice using the same steps described above. After the third round of panning the target binding of selected phages was tested by ELISA. After the third round of panning individual clones were picked up from plates and grown in 96-deepwell plates (Eppendorf, Hamburg, Germany). Phages were isolated from the culture supernatant using PEG precipitation. 96-well plates were coated with the target mAbs as in case of the selection.
• the isolated phages were diluted in TBS-T ween and incubated for 1 hour at room temperature in the antibody coated microtiter wells. Bound phages were detected with anti- Mi 3 antibody-horseradish peroxidase conjugate using OPD substrate. Results were scanned with a Victor 2 (PerkinElmer/Wallac, Waltham, MA) microplate reader. ELISA positive clones were selected for DNA sequencing. Phage clones with confirmed binding (ELISA) to the target mAbs were grown in deepwell plates and isolated by PEG precipitation. Single stranded DNA was prepared from them using precipitation with 4 M NaI and ethanol. The sequencing was done by Biomi Ltd.
• EIS epitope redundancy score
• EIS ⁇ (Xi j)/n*m, where Xi,j is the calculated identity score between the ith sequence from the first dataset and the jth sequence from the second dataset; m and n are the number of unique sequences in the two datasets.
• Immunoprecipitation and SDS PAGE electrophoresis was used to specifically precipitate and isolate cognate protein antigens for the mABs. Specific bands were cut from the gels dugested with trypsin and analyzed with MS/MS methodolgy.
• LRR leucine -rich repeat
• Leucine comprises 66 of the 312 amino acids, and LRGl contains at least 8 24-amino acid leucine -rich repeats.
• Increased LRGl expression was detected in GCSF-treated human cells derived from a patient with myeloproliferative disorder. In contrast, decreased LRGl expression was detected after PMA treatment and induction of monocytic differentiation of HL-60 cells.
• LRGl leucine rich alpha-2 glycoprotein
• the invention discloses novel antibodies that recognize specific epitopes in LRGl that represent cancer-specific biomarkers.
• BsiO392 is an IgG type monoclonal antibody.
• the heavy chain variable region amino acid sequence is represented in SEQ ID NO: 92 (see Figure 8), which is reproduced below (CDRs are underlined):
• Fig 9a The difference in biomarker level with BsiO392 is represented Fig 9a, showing a very substantial difference between control and lung cancer.
• BsiO392 recognizes a 4OkDa band in total control plasma samples (see fig 9b).
• fig 9b By mass spectrometry analysis of the antigen, various peptide sequences were obtained (see table below), which demonstrate that BsiO392 binds to LRGl.
• BsiO352 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 89 (see figure 8), which is reproduced below (CDRs are underlined):
• biomarker level with BsiO352 is represented Fig 10, showing a very substantial difference between control and lung cancer.
• BsiO352 recognizes a 4OkDa band in total control plasma samples similar to BsiO392.
• mass spectrometry analysis of the antigen various peptide sequences were obtained (see table below), which demonstrate that BsiO352 binds to LRGl .
• YLFLNGNK Peptides bound by BsiO352 have been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 26-35.
• BsiO351 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 88 (see fig 8), reproduced below (CDRs underlined).
• BsiO351 The difference in biomarker level with BsiO351 is represented Fig 11, showing a very substantial difference between control and lung cancer.
• BsiO351 recognizes a 4OkDa band in total control plasma samples similar to BsiO392.
• mass spectrometry analysis of the antigen two peptide sequences were obtained (KDLLLPQPDLRY and RTLDLGENQLETLPPDLLR), which demonstrate that BsiO351 binds to LRGl .
• Bsi358 BsiO358 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 90 (see fig 8) reproduced below (CDRs underlined).
• Fig 12a The difference in biomarker level with BsiO358 is represented Fig 12a, showing a very substantial difference between control and lung cancer.
• Western blots with BSI0358 on normal and LC plasma samples before and after depletion of abundant proteins by Agilent depletion columns is represented Fig 12b.
• Molecular mass of reactive band from LC plasma seems to be lower, suggesting that BSI 0358 reacts with a disease-specific form of protein as well.
• Peptides bound by BsiO358 have been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 1-13.
• BsiO358 can image non small cell lung cancer (NSCLC), as illustrated fig 12c: >90% of NSCLC is positive and 17.6% of SCLC is positive.
• BsiO359 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 91 (see fig 8) reproduced below (CDRs)
• VTVSA The difference in biomarker level with BsiO359 is represented Fig 13a, showing a very substantial difference between control and lung cancer.
• BsiO358 BsiO358 alone or in combination, detects a Lung Cancer specific form of the cognate antigen (see Figure 13b).
• BsiO359 binds to purified natural Alpha- 1 -anti chymotrypsin and works in SW assays with antibodies that recognize Alpha- 1 -antichymo trypsin.
• Peptides bound by BsiO359 have been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 39-42.
• BsiO359 can be used efficiently for cancer histopathology: staining was positive for 84% of NSCLC, 88% adenocarcinoma, 64% squamosus cell carcinoma, and 100% of large cell carcinomas. BsiO359 thus could be useful for imaging.
• Complement factor 9-binding antibodies is a component of the complement system, a multi -protein biochemical cascade which aids to clear pathogens. The cascade is activated upon binding of IgG or IgM to pathogen molecules. C9 is one of the terminal components of the cascade and is responsible for forming pores in target cells leading to their destruction. Deficiencies in complement proteins are believed to be linked to auto-immunity and higher sensitivity to infections.
• the cDNA coding for C9 was sequenced and the protein sequence—537 amino acids in a single polypeptide chain—was derived. The amino -terminal half of C9 is predominantly hydrophilic and the carboxyl-terminal half is more hydrophobic.
• the amphipathic organization of the primary structure is consistent with the known potential of polymerized C9 to penetrate lipid bilayers and cause the formation of transmembrane channels as part of the lytic action of MAC.
• Marazziti et al. (1988) compared gene and protein structure of C9 and compared both with low density lipoprotein receptor (606945).
• the C9 gene is composed of 11 exons with lengths between 100 and 250 bp, except for exon 11 which extends over more than 1 kb, as it includes the 3-prime untranslated region.
• Witzel-Schlomp et al. gave revised information on the structure of the C9 gene, especially the exon-intron boundaries
• C9 has not been associated with cancer of any type.
• the present invention discloses specific antibodies against C9 that can be used as lung cancer biomarkers.
• BsiO272 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 86 (see fig 8) reproduced below (CDRs).
• Fig 14a The difference in biomarker level with BsiO272 is represented Fig 14a, showing a very substantial difference between control and lung cancer.
• BsiO272 recognizes a band in total control plasma samples which is compatible to a 559aa polypeptide corresponding to mature C9 polypeptide (Fig 14b).
• Fig 14b By mass spectrometry analysis of the antigen, several peptide sequences were obtained (see table below), which demonstrate that BsiO272 binds to C9.
• Peptides bound by BsiO272 have been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 14-25.
• BsiO272 stains non small cell lung cancer (NSCLC), as illustrated fig 14c: >74% of NSCLC is positive.
• Haptoglobin (HP), NM 005143 is a tetrameric protein that functions to bind free plasma hemoglobin, thereby allowing degradative enzymes to gain access to the hemoglobin, while at the same time preventing loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin. Mutations in the HP gene and/or its regulatory regions cause ahaptoglobinemia or hypohaptoglobinemia.
• Hp haptoglobin
• Bsi0033 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 77 (see fig 8) reproduced below (CDRs underlined).
• Peptides bound by Bsi0033 have also been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 36- 38.
• Bsi0033 can react with non small cell lung cancer (NSCLC) as well as large cell carcinomas: 81% of NSCLC, 89% adenocarcinoma, 20% squamosus cell carcinoma, and 83% of large cell carcinomas are positive.
• NSCLC non small cell lung cancer
• Bsi0071 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 81 (see fig 8) reproduced below (CDRs underlined).
• biomarker level with Bsi0071 is represented Fig 16a, showing a very substantial difference between control and lung cancer.
• Bsi0071 recognizes a band which is consistent with reaction of HP under non-reducing conditions, similar to BsiOO33.
• mass spectrometry analysis of the antigen several peptide sequences were obtained (see table below), which demonstrate that Bsi0071 binds to HP and HRP.
• Complement Factor H (NM OOO 186) is a member of the Regulator of Complement Activation (RCA) gene cluster.
• the CFH protein contains twenty short consensus repeat (SCR) domains, is secreted into the bloodstream, and has an essential role in the regulation of complement activation, restricting this innate defense mechanism to microbial infections. Mutations in this gene have been associated with hemolytic-uremic syndrome (HUS) and chronic hypocomplementemic nephropathy.
• Complement factor H (CFH) is an inhibitor of the alternative complement pathway.
• the present invention discloses specific antibodies against CFH that can be used as lung cancer biomarkers.
• Bsi0077 is an IgG type monoclonal antibody.
• the heavy chain variable amino acid sequence is represented in SEQ ID NO: 83 (see fig 8) reproduced below (CDRs).
• NSCLC non small cell lung cancer
• BsiO271 is an IgG type monoclonal antibody. Antigen binding was determined by western blot and MS analysis. By mass spectrometry analysis of the antigen, several peptide sequences were obtained (see table below), which demonstrate that Bsi0077 binds to CFH.
• R.TGESVEFVCK.R Peptides bound by BsiO271 have been identified and verified, using either phage display technique or direct Elisa binding. These peptides are presented as SEQ ID NOs: 43-56.
• Bsi0077 can image non small cell lung cancer (NSCLC) as well as carcinomas: 81% of NSCLC, 84% adenocarcinoma, 60% squamous cell carcinoma, and 67% of large cell carcinomas were positive.
• NSCLC non small cell lung cancer
• the best linear model was calculated using leave-one out cross-validation on a training set, and the accuracy was estimated on a test set of independent samples.
• the accuracy of the 1013 combinations is plotted as a histogram where the number of panels with specific accuracy is reported on the y-axis (see Fig 18). The number of the occurrences of each antibody in the panels with accuracy higher than 0.8 is plotted on the y-axis.
• the optimal threshold (0.115) was calculated from the logistic regression model to provide the optimum combination of sensitivity (80.4%) and specificity (86.2 %).
• the performance of this classifier to predict patients with different stages of the disease show that its performance increases with the stage of the disease, and even at stage I the sensitivity is 77.3%. Slightly better performance of the classifier should be also noted for patients with squamous cell carcinoma (Table II).
• Adenocarcinoma 66 (85) 77.6 %
• ROC curves are presented in Figure 20 where panels are numbered from 1-10 based on this table.
• the results show the antibodies of the invention differentiate control and lung cancer, either alone or in combinations. These antibodies allow the design of efficient diagnostic products (e.g., devices, kits) to detect, monitor or image cancer in human subjects.
• the epitope redundancy is expressed as percentage of identical residues in the aligned set of peptide sequences between two antibodies (see Methods).
• the functional redundancy is expressed as the adjusted chi-square of fitting the correlation of the responses of two antibodies with the 610 clinical samples measured in the HT-ELISA screening to a linear function (see
• Peptides bound by further antibodies of the invention are Peptides bound by further antibodies of the invention. % represent peptide sequence occurrence in the tested group.

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