**8. Enhancement of chemodrug inhibition of tumor by antibody**

Our P1G4 antibody was found to possess a clinically useful property. Using eAGR2+ pancreatic PDX in mice, the combination of pancreatic cancer drug Gemcitabine (Gem) and P1G4 (P1) reduced tumor growth compared to Gem alone. This difference was statistically significant (*P* < 0.05) [22]. Immunostaining for Ki67 indicated less (AGR2+ ) tumor cell proliferation in P1 + Gem, which was manifested by the size of the corresponding resected tumors (**Figure 7**). ELISA indicated that serum AGR2 levels could be correlated with tumor burden. Once Gem was discontinued, the tumors in the Gem-only group relapsed immediately, and grew at a faster rate than those in the P1 + Gem group despite an effectively reduced antibody concentration as antibody treatment was terminated after 28 days. The combination of P3A5 (P3) and Gem showed no such enhancement. Parenthetically, our data also confirmed that Agr2 was not secreted from normal mouse organs into blood. The

#### **Figure 6.**

*Tumor localization of P3A5. The SPECT/CT scans show specific uptake of radiolabeled P3A5 by Agr2+ DT606 tumors (\*) in two C57BL/6 mice.*

#### **Figure 7.**

*Drug inhibition of tumor growth enhancement by P1G4. (A) Representative immunohistochemistry images show the effect (from top to bottom) of IgG control, P1G4 (P1) alone, Gemcitabine (GEM) alone, P1 + GEM. Ki67 staining indicates that tumors treated with drug still had high proliferation rate, which was limited in P1 + GEM tumors. CD3 shows T cell infiltration in the GEM and P1 + GEM groups. (B) Tumors resected at week 6 from the different treatment groups are compared. The smallest size is found in the P1 + GEM group (arrowed).*

internal organs of liver, spleen, stomach, intestine, colon, and pancreas were histologically examined, and no visible pathologic changes were identified. The sparing of organs in anti-AGR2 tumor targeting was also reported by another group [46]. The mechanism behind this epitope-dependent phenomenon is unknown but could be related to a reported observation of increased tumor inhibition by an antibody to AGR3 in combination with the chemodrug cisplatin [47]. AGR3 is a close family member of AGR2. Both AGR2 and AGR3 tend to be elevated in cancer, though to different levels as found in prostate cancer [10]. The combination of monoclonal antibody plus biological inhibitors are being pursued to treat more successfully non-small cell lung cancer [48].

#### **9. Tumor cell lysis in vitro**

In our earlier work, 51Cr radiolabeled target cancer cells were exposed to TAA antibodies and human serum (as a source of complement factors) or peripheral blood leukocytes [43, 49]. By CDC, the chimeric antibody generated higher cytotoxicity at all complement dilutions. By ADCC, at a ratio of 100:1 blood leukocytes to target cells, the chimeric lysed a greater fraction of the cancer cells and gave 50% cytolysis at 100-fold lower concentration than the mouse antibody. ADCC was observed at a 3:1 ratio of effector to target cells when the chimeric (at 2.5 μg/ml) was used. Cell killing was specific because ADCC was not observed with cell lines lacking the target antigen.

To test the anti-tumor effect of chimeric antibodies, we incubated PC3 prostate cancer cell line in the presence of donated human serum. Like pancreatic cancer cells, cell surface expression of eAGR2 was found on PC3 cells. Spin-concentrated chimeric IgG was used with human serum for CDC. PC3 cells were incubated with freshly donated human serum and added AGR2 antibodies. There was no observable effect on cell viability in the culture well with human serum only, as was the well with mouse P3A5 + serum. In the well with a cocktail of chimeric IgG1, IgG2, and IgG4, cell growth was inhibited with well surface showing areas devoid of cells,

**105**

**Figure 8.**

*conjugate (in ng/ml, x-axis).*

*Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2*

and clusters of pyknotic cells in suspension [22]. We postulate that a combination of IgG subtypes would be more effective than IgG1 alone since our normal immune response produces these IgG types, each exhibiting a unique profile with respect to immune complex formation, complement activation, recruitment of effector cells, and half-life [50]. For example, strong antitumor activities were observed for an IgG3 antibody targeting a melanoma-associated ganglioside [51]. We did not obtain chimeric IgG3 for our monoclonal antibodies. We will attempt to clone Cγ3 from a larger volume of blood or a pool of several donations using γ3-specific primer oligonucleotides. Our cloned human Cγ and Cκ can accept any new VH and VL of

In addition to antibodies that rely on immune system components, a cytotoxic drug can be linked directly to the AGR2 antibodies to produce an antibody-drug conjugate (ADC). ADC delivers the drug payload to the target organs or tissues [52]. For anti-AGR2 ADC, drug compounds were constructed by covalently linking poly(N-isopropylacrylamide) to both P1G4 and P3A5 via carbodiimide chemistry. The linking polymer was synthesized by reversible addition fragmentation chain transfer (RAFT) with a carboxylate chain end [53]. To conjugate antibodies, the carboxylate was converted to an active ester for formation of an amide bond to lysine residues [53–56]. We have also developed a block copolymer with tetrafluorophenyl (TFP) ester monomers to drive the antibody conjugation [57]. The resulting anti-AGR2 ADC were confirmed by gel electrophoresis, which showed the larger molecular weight products compared to the unconjugated antibodies (**Figure 8**). After the size-exclusion chromatography, the purified ADC were shown by ELISA to bind AGR2 (**Figure 8**). Sample solutions containing a constant AGR2 concentration were mixed with ADC from 125 to 1,000 ng/ml. ELISA measured the unbound AGR2 with higher ADC concentrations resulting in less free AGR2 in the solution. Thus, our conjugation chemistry did not affect appreciably the antigen binding affinity of the antibodies. Polymer chains with improved loading capacity by incorporating functional groups [58] could be developed for conjugation to docetaxel, doxorubicin, and other drugs. To improve delivery efficiency, the polymer can be engineered to increase circulation time,

*Antibody drug conjugate. The left panel shows the conjugated products (lane 1) vs. unconjugated antibody (lane 4). The right plot shows more AGR2 bound (percentage, y-axis) with higher ADC concentrations of the* 

*DOI: http://dx.doi.org/10.5772/intechopen.96492*

antibodies developed against novel TAA.

and the polymer composition can be modulated.

**10. Antibody-drug conjugation**

*Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2 DOI: http://dx.doi.org/10.5772/intechopen.96492*

and clusters of pyknotic cells in suspension [22]. We postulate that a combination of IgG subtypes would be more effective than IgG1 alone since our normal immune response produces these IgG types, each exhibiting a unique profile with respect to immune complex formation, complement activation, recruitment of effector cells, and half-life [50]. For example, strong antitumor activities were observed for an IgG3 antibody targeting a melanoma-associated ganglioside [51]. We did not obtain chimeric IgG3 for our monoclonal antibodies. We will attempt to clone Cγ3 from a larger volume of blood or a pool of several donations using γ3-specific primer oligonucleotides. Our cloned human Cγ and Cκ can accept any new VH and VL of antibodies developed against novel TAA.
