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PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo., Competing Interests: H.L., A.M.S., L.A. and U.E. have submitted a patent application (Methods and compositions for PDGF-CC inhibition, PCT/US2017/040170) on these antibodies. A.M.S. and U.E. are shareholders of a company (Paracrine Therapeutics AB), developing these antibodies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Periostin (POSTN), a secreted homodimeric protein that binds integrins αvβ3, αvβ5, and α6β4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvβ3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status., (© 2015 UICC.)

The immunocytochemical demonstration, using monoclonal antibodies, of diverse cellular constituents has improved our understanding of many aspects of oncology. This review will focus on this approach to facilitate the detection of human tumors, improve their histological classification and provide functional parameters with potential aetiological, prognostic and/or therapeutic value.

Mouse monoclonal antibodies reacting with human mammary gland constituents have been generated and characterized in an attempt to raise breast- or breast-cancer-specific antisera. The immunogens used in these studies included fractionated human milk fat globule membrane, the human breast cancer cell line MCF7 and a crude membrane preparation derived from an axillary nodal metastasis from a patient with breast cancer. Of the antibodies obtained, 8 were characterized and found to bind to different structures in the normal breast. The antibody LICR-LON-LC28 recognizes secretory component and binds strongly to normal resting and lactating breast, but only focally to a minority of breast carcinomas. The antibodies LICR-LON-14.1 and 32.2 react strongly with the lactating breast and recognize kappa- and beta-casein, respectively. Caseins are not produced by breast tumors. The antibodies LICR-LON-TW19.5, H10A and 39.8 all react with carbohydrate epitopes and bind heterogeneously to normal resting breast luminal epithelium and cellular subsets of breast carcinomas. LICR-LON-59.2 and 19.2 react with normal breast myoepithelial cells and the basement membrane, respectively. LICR-LON-59.2 is unusual as a myoepithelial marker in that it stains cells in the majority of breast carcinomas. LICR-LON-19.2 shows extensive reactivity to tumor cell lines in culture but has no reactivity with carcinoma cells from breast biopsies.

We have devised a method utilizing a monoclonal antibody-toxin conjugate (LICR-LON-Fib75/abrin A-chain) for ridding bone marrow of infiltrating breast cancer cells to rescue patients with autologous bone marrow following high dose therapy. Initially we examined the activity of this conjugate in vitro. Five of seven human breast cancer cell lines were killed following exposure at 10(-8) M for 2 h; this concentration only reduced bone marrow colony formation to 83% (range, 50-100%) of control bone marrow. We then examined the pattern of bone marrow recovery after high dose melphalan (200 mg/m2) in patients with advanced breast cancer who were in remission following combination chemotherapy. To do this we compared the time of recovery of the blood count in three patients who received treated marrow and seven who received untreated marrow. Mean time to recovery of the peripheral white count (greater than 1.5 X 10(9)/liter) was 16.7 days (treated) and 18.3 days (untreated), respectively. Mean time to recovery of peripheral platelet count (greater than 50 X 10(9)/liter) was 23.7 days (treated) and 18.9 days (untreated), respectively. Patients continued in remission for 1-greater than 14 mo after high dose melphalan, and remission duration was similar in patients who received treated (6.2 mo) and untreated (7.3 mo) bone marrow. These findings indicate that treatment of bone marrow with LICR-LON-Fib75/abrin A-chain conjugate does not significantly impair bone marrow recovery, and it is, therefore, possible to rescue breast cancer patients with bone marrow that has been cleansed of infiltrating cancer cells. This may have an application in patients with poor-risk primary breast cancer who have micrometastases and who may benefit from intensive therapy, but it has minimal application in patients with more advanced disease.

Present methods and systems for the generation of human monoclonal antibodies are briefly reviewed. The specificities of the available reagents are outlined. It would appear that the generation using hybridoma methods of human monoclonal antibodies to human tumor cell surface antigens is a rare event and that methods of in vitro immunostimulation may have to be used if such antibodies are to be reliably produced.

Immune-suppressed mice carrying xenografts of several different types of human germ-cell tumours were injected with a radiolabelled monoclonal antibody (LICR LON/HT13) raised against membrane components of a human germ-cell tumour (HX39). Subsequent assessment of radioactivity in excised organs and tumours showed a selective accretion of antibody in the tumour. Quantitative autoradiography supported the results of radiolocalization observed in vivo in different tumours, and also showed that the antibody localized to viable tumour cells and in close association with their cell membrane. The vascular architecture of tumours was found to be an important factor governing antibody distribution. No localization occurred with radiolabelled normal mouse IgG.

A range of primary and metastatic human breast carcinomas has been examined with respect to the staining by four monoclonal antibodies which were raised to the human milk fat globule membrane. Within the normal breast the luminal epithelial cells expressing the antigens detected by the monoclonal antibodies were heterogeneous in their distribution. The heterogeneity was not only confined to single cells, but also to regions within the breast. The breast carcinomas also expressed the antigens in a variable manner. Morphological differentiation and functional differentiation, defined by the monoclonal antibodies, were not invariably coincident. Lymph node metastases gave similar results to the primary carcinomas. The monoclonal antibodies have revealed a heterogeneity, with respect to surface antigenic expression, within the normal and neoplastic breast epithelium. This cellular heterogeneity of breast carcinomas, may have significant prognostic and therapeutic implications in the management of primary breast cancer.

The ability of a radiolabelled monoclonal antibody, LICR-LON-M8 (M8), to locate metastatic breast carcinomas has been investigated. The scans generated by M8, either when labelled with radioiodine, or when conjugated with diethylenetriamine-pentaacetic acid (DTPA) and labelled with radioactive indium (111In), have been compared with X-rays and 99mTc-methyl diphosphonate (MDP) bone scans. All 10 patients with skeletal metastases had positive 111In-DTPA-M8 scans and the overall correlation with X-rays and MDP scans was good but varied with the region studied. By contrast, radioiodinated M8 did not detect metastases at any site. The discrepancies between 111In-DTPA-M8 images and conventional techniques may be related to the different stages in the evolution and development of the lesion at which the various techniques detect bone metastases.

Mouse monoclonal antibodies have been raised to the human milk fat globule membrane. The distribution of the antigens detected by four of the antibodies has been examined in formalin-fixed paraffin-embedded human tissues by light microscopic immunocytochemistry. The four antibodies stain lactating breast and normal resting breast. Two exclusively stain the luminal membranes of breast epithelial cells. A third antibody stains in addition the lateral membranes of duct epithelial cells. The fourth antibody stains both epithelial and myoepithelial cells. None of the antibodies is breast specific, nor do they stain every epithelial cell within the breast. Instead, each antibody reveals a complex and heterogeneous distribution of staining throughout the normal tissues. Within the breast, the staining by a given antibody is usually segmental and conforms to secretory units and their associated ducts. Similarly heterogeneous patterns of staining are also observed in the extramammary normal tissues. Despite the apparent morphological identity between breast epithelial cells when examined by conventional light microscopy, the hitherto unrecognised "functional" heterogeneity, which has been revealed by the monoclonal antibodies could have importance in understanding the biology of the normal breast and the pathology of breast cancer.

Monoclonal antibodies were raised against the human germ-cell tumor line, LICR LON HT39/7. The cells of this human embryonal carcinoma cell line were first fixed in formol saline and then dehydrated prior to immunization so that antibodies were produced which reacted with antigens surviving conventional histological processing. Eleven hybrid clones secreted antibody that reacted with both formol saline-fixed and living cells of the immunogen. One of these antibodies, LICR LON FC 10.2, has been found to react with a membrane glycoprotein of molecular weight 200,000 restricted in distribution to the surfaces of undifferentiated teratoma-derived cell lines, the luminal surface of human fetal intestine and bronchus and areas of putative endodermal differentiation within germ-cell neoplasms.

A variety of monoclonal antibody probes to the human breast have been prepared by various groups of workers. These probes are revealing hitherto unrecognized biological and biochemical differences in both normal and neoplastic breast cells. Further study is needed, however, in order to understand fully the significance of these differences. In the meantime, the use of immune probes can improve our ability to detect disease at an earlier stage in sites such as the bone marrow, and may be heralding a new era in the detection and treatment of disseminated breast cancer.

A rapidly-growing, HAT-sensitive cell line LICR-LON-HMy2 has been derived from the ARH-77 human plasma cell leukemia-derived line. It lacks the enzyme hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). Hybrids have been reproducibly made for more than a year and in independent laboratories with lymphocytes from tonsils, lymph nodes and peripheral blood and tonsil lymphocytes cultured with antigens. The parent line and hybrids are very robust in culture and double in 20-30 h. Hybrids clone easily and have stable karyotypes, most with modal numbers in the sixties. Stable Ig secretion patterns have been observed over 20-30 passages, and after cloning. It was estimated that about half the hybrids produce new immunoglobulin chains in addition to the parent cell line's IgG1 (kappa light chain). High, but not limiting, density hybridoma cultures (approximately 5 x 10(5) cells/ml) typically produce 0.25-2 micrograms/ml immunoglobulin per day, but some hybrids produce more. A high-secreting variant of LICR-LON-HMy2 has been derived. The LICR-LON-HMy2 line is human and is distinct from the HAT-sensitive human B cell-lines SKO-007 and GM 1500-6TG Al 2. It is available for distribution.

The value of a monoclonal antibody, LICR-LON-Fib-75 (Fib-75), which mediates complement lysis and recognises an antigen present on all epithelial-tumour cells so far studied but not on lymphoid cells or bone-marrow stem cells, in clearing infiltrated bone marrow was assessed. Chromium-51-release and trypan-blue-exclusion assays showed that no tumour cells were viable after exposure to Fib-75 and complement except when large clumps (greater than 500 cells) were present, whereas Fib-75 had no significant effect on bone-marrow cells, as judged by colony-forming-unit and pluripotential-stem-cell assays.

This resume has focussed attention upon some aspects of the clinical and pathological utility of breast-directed antibodies. Such related probes may also be of value in novel aspects of therapy for this group of disorders and as agents for radio-immunolocalisation.

Monoclonal antibody LICR -LON- M18 identifies the immunodominant oligosaccharide sequence of the I(Ma) blood-group antigen: Gal beta 1----4GlcNAc beta 1----6--. In primary breast cancers this structure is almost totally cryptic, due to "masking" by sialic acid, but can be revealed by digestion with the specific glycosidase neuraminidase. Following desialylation, light microscopic immunohistochemical examination has revealed the epitope identified by LICR -LON- M18 to be heterogeneously distributed throughout the population of breast carcinoma cells. These tumor cells express the antigen as both a cytoplasmic and a surface membrane determinant. In the normal human breast, this structure is expressed exclusively along the luminal plasma membranes of the duct and alveolar littoral epithelial cells. Desialylation of tissue sections of normal resting and lactating breast epithelium with neuraminidase virtually abolishes the heterogeneous intercellular distribution of the I(Ma) determinant. In desialylated nonneoplastic breast tissues, the expression of this antigen is observed within the cytoplasm of some myoepithelial cells, but not in the littoral epithelial cells. The expression of the I(Ma) antigen by neoplastic and normal breast epithelial cells has also been compared with that of the oligosaccharide sequence Gal beta 1----3GalNAc. This structure, recognized by peanut agglutinin, forms the dominant portion of the Thomsen-Friedenreich antigen. With respect to normal and lactating breast epithelial cells, both oligosaccharide structures are sialylated and appear to be similarly misprocessed by breast carcinomas. The masking of surface carbohydrate determinants and the faulty processing of structures usually expressed on the surface of non-neoplastic breast epithelial cells may be important phenomena in the pathobiology of breast carcinomas.