**4. Differential expression of AGR2 in prostate cancer – primary vs. metastasis**

AGR2 in prostate cancer shows two seemingly conflicting features in tumor biology. High expression in primary tumors is linked to better patient survival. Together with cell surface CD10 (membrane metallo-endopeptidase), four cancer phenotypes can be distinguished: AGR2<sup>+</sup> CD10<sup>−</sup> , AGR2<sup>−</sup> CD10<sup>+</sup> , AGR2<sup>−</sup> CD10<sup>−</sup> , and AGR2<sup>+</sup> CD10<sup>+</sup> ; normal luminal cells are AGR2<sup>−</sup> CD10<sup>+</sup> . For high-grade disease, the AGR2<sup>+</sup> CD10<sup>−</sup> phenotype is associated with a near 10-fold survival advantage than that of AGR2<sup>−</sup> CD10<sup>+</sup> ; those of AGR2<sup>−</sup> CD10<sup>−</sup> and AGR2<sup>+</sup> CD10<sup>+</sup> in between [27]. Not unexpectedly, a majority of these tumors were typed AGR2<sup>−</sup> CD10<sup>+</sup> (**Figure 2**). It appears that CD10 plays an important role in the extraprostatic

**99**

are CD10<sup>+</sup>

*show a contrasting trend.*

**Figure 2.**

with the AGR2+

noma (AGR2−

CD10−

CD10−

of these metastases are also AGR2+

AGR2<sup>−</sup>

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

spread of cancer cells to local lymph nodes because cancer cells in involved

*AGR2 expression and patient survival. Left: the plot shows the outcome of high-stage patients grouped by AGR2 and CD10. At 5 years, 85% of AGR2hiCD10lo were recurrence-free compared with just 25% of AGR2loCD10hi. Right: survival analyses for prostate cancer (top) and lung cancer (bottom) patients (<65y)* 

derived cancer cell line LNCaP and patient-derived xenograft (PDX) LuCaP 35

ization of CD10 to cell interior in cancer cells of higher grades where it appears to interact with cytosolic heat shock proteins [34]. As in the case for AGR2, CD10 has extracellular eCD10 and intracellular iCD10, but in this case, iCD10 is specific to cancer cells. This suggests that protein trafficking is abnormal in cancer cells. AGR2 expression is also associated with prostate cancer differentiation, with Gleason grade 3 (well-differentiated) cancer cells showing a 10-fold higher level than Gleason grade 4 (less differentiated) cancer cells [10]. Its association with lower tumor grade predicts better survival for patients harboring AGR2<sup>+</sup> tumors [11] (**Figure 2**). Similarly, in breast cancer AGR2 is associated with better survival [35]. In contrast to AGR2, CD10 is more prominent in higher grade prostate tumors, and is associated with poorer survival for patients [33, 36]. However, most of the distal bone and soft tissue metastases contain cancer cells

role of AGR2 in cancer spread as we reported for pancreatic cancer [37]. Possible mechanisms on how AGR2 promotes metastasis include disruption of epithelial cell adhesion, imparting invasive behavior to tumor cells [19], inducing apoptosis in susceptible normal cells by secreted AGR2 to undergo apoptosis [38], and through activating matrix metalloproteases, cathepsins B and D [37], which could facilitate tumor cells access to the circulatory system. Importantly, inhibition of AGR2 expres-

sion in lung cancer cells leads to their loss of metastatic capability [19].

(AGR2−/lo) [33]. Node metastasis-

AGR2lo, respectively. A notable feature is the local-

phenotype, the exception being tumors of small cell carci-

) [27], (**Figure 3**). PDX LuCaP lines established from samplings

[27]. These results underscore the important

regional lymph nodes are invariably CD10<sup>+</sup>

and CD10<sup>+</sup>

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

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

#### **Figure 2.**

*Advances in Precision Medicine Oncology*

by DNA microarray analysis of CD9+

and CD26+

next to the iAGR2+

by normal iAGR2+

contrast, we found that AGR2+

was a strong correlation, *R*<sup>2</sup>

CD10<sup>+</sup>

CD10<sup>−</sup>

than that of AGR2<sup>−</sup>

**metastasis**

and AGR2<sup>+</sup>

the AGR2<sup>+</sup>

urothelial cells (signal value = 105.54) [25]

prostate cancer cells (signal value = 4168.03) [10]. A previous study

urothelium [18] (**Figure 1**, bottom panel) or benign glands of

estimated that adenocarcinoma cells had 80% iAGR2 and 20% eAGR2 while in all non-tumor cells AGR2 was located intracellularly [19]. Faint AGR2 staining of the stroma surrounding prostate tumor glands could be evidence of AGR2 secretion [27] (**Figure 1**, top panel). No such staining was detectable in the lamina propria

the prostate. In prostate cancer cells, AGR2 could be activated by ER stress with down-regulation of unfolded protein response genes leading to an accumulation of

The contrasting localization of eAGR2 and iAGR2 is well illustrated by the bladder. The entire urothelium is positive for AGR2 expression as shown by immunostaining [18] (**Figure 1**). If AGR2 is secreted, then a substantial amount of this protein would be found in voided urine. In fact, little AGR2 is released

supported by urine proteome database queries that AGR2 was not listed in the *UrinePA-PeptideAtlas* of 2,500 proteins profiled by large-scale proteomics [29], and in the core urinary proteome of 587 proteins obtained from healthy people [30]. In

bladder cancer patient was scored 7.5-fold higher than buffer control for AGR2. Five of 20 bladder cancer patient urine in one cohort were tested positive (AUC = 0.75), which matched the 25% bladder cancer being positive for AGR2 expression [18]. Our urine assay also detected AGR2 secreted by prostate cancer producing a similar AUC [31]. For serum, level of AGR2 is near background in healthy people [32]. Query of the *PeptideAtlas* database yielded very low AGR2 signature peptide counts. However, when sera of five prostate cancer patients were analyzed for AGR2, there

those of PSA (in ng/ml) [32]. This result would not be possible if there was a basal

such as the urothelium and lung epithelium into the circulatory system [32].

**4. Differential expression of AGR2 in prostate cancer – primary vs.** 

; normal luminal cells are AGR2<sup>−</sup>

; those of AGR2<sup>−</sup>

[27]. Not unexpectedly, a majority of these tumors were typed AGR2<sup>−</sup>

(**Figure 2**). It appears that CD10 plays an important role in the extraprostatic

AGR2 in prostate cancer shows two seemingly conflicting features in tumor biology. High expression in primary tumors is linked to better patient survival. Together with cell surface CD10 (membrane metallo-endopeptidase), four can-

CD10<sup>−</sup>

CD10<sup>−</sup>

phenotype is associated with a near 10-fold survival advantage

, AGR2<sup>−</sup>

and AGR2<sup>+</sup>

CD10<sup>+</sup>

CD10<sup>+</sup>

CD10<sup>+</sup>

level of blood AGR2 arising from possible secretion by AGR2+

cer phenotypes can be distinguished: AGR2<sup>+</sup>

CD10<sup>+</sup>

urothelial cells as detected by ELISA [18]. This result was

bladder cancer cells secreted AGR2. Urine from a

= 0.93, found between levels of AGR2 (in pg/ml) and

cells of normal tissue

, AGR2<sup>−</sup>

. For high-grade disease,

in between

CD10<sup>+</sup>

CD10<sup>−</sup> ,

In summary, three AGR2 expression patterns have been described in solid tumors: (1) iAGR2 positive for normal urothelial cells, eAGR2 positive for 25% primary tumors [18]; (2) no AGR2 for prostate epithelial cells, eAGR2 positive for >95% primary tumors [10], similar to pancreas [12] and breast [13]; (3) iAGR2 positive for normal bronchial epithelial cells, eAGR2 positive for>95% non-small cell lung tumors [14].

misfolded proteins like that in pancreatic cancers cells [28].

**3. Differential subcellular localization of AGR2**

**98**

*AGR2 expression and patient survival. Left: the plot shows the outcome of high-stage patients grouped by AGR2 and CD10. At 5 years, 85% of AGR2hiCD10lo were recurrence-free compared with just 25% of AGR2loCD10hi. Right: survival analyses for prostate cancer (top) and lung cancer (bottom) patients (<65y) show a contrasting trend.*

spread of cancer cells to local lymph nodes because cancer cells in involved regional lymph nodes are invariably CD10<sup>+</sup> (AGR2−/lo) [33]. Node metastasisderived cancer cell line LNCaP and patient-derived xenograft (PDX) LuCaP 35 are CD10<sup>+</sup> AGR2<sup>−</sup> and CD10<sup>+</sup> AGR2lo, respectively. A notable feature is the localization of CD10 to cell interior in cancer cells of higher grades where it appears to interact with cytosolic heat shock proteins [34]. As in the case for AGR2, CD10 has extracellular eCD10 and intracellular iCD10, but in this case, iCD10 is specific to cancer cells. This suggests that protein trafficking is abnormal in cancer cells. AGR2 expression is also associated with prostate cancer differentiation, with Gleason grade 3 (well-differentiated) cancer cells showing a 10-fold higher level than Gleason grade 4 (less differentiated) cancer cells [10]. Its association with lower tumor grade predicts better survival for patients harboring AGR2<sup>+</sup> tumors [11] (**Figure 2**). Similarly, in breast cancer AGR2 is associated with better survival [35]. In contrast to AGR2, CD10 is more prominent in higher grade prostate tumors, and is associated with poorer survival for patients [33, 36].

However, most of the distal bone and soft tissue metastases contain cancer cells with the AGR2+ CD10− phenotype, the exception being tumors of small cell carcinoma (AGR2− CD10− ) [27], (**Figure 3**). PDX LuCaP lines established from samplings of these metastases are also AGR2+ [27]. These results underscore the important role of AGR2 in cancer spread as we reported for pancreatic cancer [37]. Possible mechanisms on how AGR2 promotes metastasis include disruption of epithelial cell adhesion, imparting invasive behavior to tumor cells [19], inducing apoptosis in susceptible normal cells by secreted AGR2 to undergo apoptosis [38], and through activating matrix metalloproteases, cathepsins B and D [37], which could facilitate tumor cells access to the circulatory system. Importantly, inhibition of AGR2 expression in lung cancer cells leads to their loss of metastatic capability [19].

#### **Figure 3.**

*AGR2 in prostate cancer metastases. Tumor cells in distal metastases – bone (top left), liver (top right), lymph node (bottom left) – are strongly stained for AGR2. A metastasis containing small cell carcinoma (bottom right) is not stained.*

## **5. AGR2 in bladder cancer and lung cancer**

Although 25% primary bladder tumors of a study cohort were found positive for AGR2, this percentage increased to 45% in the synchronous lymph node metastases [18]. The discordance between primary cancer and lymph node metastasis could involve a phenotypic change [39], which could occur in bladder cancer cells going from AGR2− to AGR2+ . AGR2 in bladder cancer, unlike prostate cancer, showed no link to patient survival in one cohort analyzed [18]. A role for CD10 in lymph node spread of bladder cancer was also not apparent [18]. Recently, UW Urology initiated the bladder cancer rapid autopsy program modeled on the Department's success in obtaining>40 prostate cancer LuCaP lines with various characteristics [40], and has established lines, dubbed CoCaB, from primary and metastatic urothelial cancer. To date, seven lines are available: CoCaB 1, CoCaB 8 urothelial carcinoma, CoCaB 11, CoCaB 19 squamous carcinoma, CoCaB 12 urothelial with sarcomatoid component; three lines from metastases of deceased patients: CoCaB 10 (liver metastasis) urothelial carcinoma, CoCaB 14.1 (omentum metastasis) and CoCaB 14.2 (liver metastasis) urothelial carcinoma with squamous features. These lines were profiled by RNAseq and exome sequencing. Notably, the lines derived from metastases showed high AGR2 expression (**Figure 4**). This is in line with our result on prostate cancer metastases where, except for small cell carcinoma, all tumors as well as the LuCaP lines derived from metastases showed high AGR2 expression [27]. Likewise, pancreatic cancer metastases invariably showed high AGR2 expression [37].

In non-small cell lung cancer, higher AGR2 expression in primary tumors is associated with a poorer outcome for patients under 65 [14, 19], though over 90% of the tumors are AGR2+ (**Figure 2**). This is in contrast to the finding for prostate cancer. Thus, depending on the microenvironment, the metastatic function of AGR2 is not predominant in differentiated prostate tumors. When cancer cells have escaped the prostatic capsule (via CD10), AGR2 appears to be essential for distal

**101**

to be elucidated.

**Figure 4.**

*small red oval.*

against 0.7% of AGR2−

**6. Chimeric human:mouse AGR2 antibodies**

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

colonization of other organs for prostate cancer cells. In the disease course, AGR2 expression could be down- or up-regulated in cancer cells through mechanisms yet

*Bladder cancer cell types. Cancer cell lines are identified on the x-axis (CoCaB and uc). Histogram represents fold in expression difference as scored by RNAseq signal values on the y-axis for MME/CD10, AGR2, XIST (X-inactive specific transcript) and CD24. The two CoCaB lines established from metastases are indicated by* 

A meta-analysis of the published data on the clinical utility of AGR2 expression in various solid tumors summarizes the link to survival findings [41]. AGR2 expression in primary cancer can be associated with better survival for prostate cancer, no survival advantage in bladder cancer, poorer survival for lung cancer. While AGR2 in pancreatic cancer could have a significant impact in treating both early and advanced diseases, anti-AGR2 therapy in prostate cancer could prove promising in treating advanced disease as 96.4% metastatic lesions of adenocarcinoma being AGR2+

Mouse monoclonal antibodies to AGR2 were generated by inoculating bacterially produced recombinant AGR2 in RBF/DnJ mice [20]. Two of eleven clones collected, P1G4 (mIgG1) and P3A5 (mIgG2a), were tested positive for binding to native AGR2. These antibodies perform well in immunostaining of frozen tissue sections but not formalin-fixed tissue. ELISA based on these two antibodies was able to detect 17 pg of cancer-secreted AGR2 in 100 μg of total urinary protein

Based on our published design [43], the mouse antibodies were converted to chimeric human:mouse. The mouse variable VH and Vκ of P3A5 and P1G4 sequences were determined via reverse transcriptase-polymerase chain reaction (RT-PCR) with designed primers from mIg mRNA isolated from the respective hybridoma cell lines. The V sequences were-matched to known murine Ighv and Igkv genes. The human constant Cγ and Cκ domain cDNA were cloned by using designed primers from white blood cells of healthy donors. The hIg cDNA were verified by DNA sequencing and restriction enzyme digestion. The Cγ (digested by *Apa* I and *Bam* HI) and Cκ (*Hind* III and *Avr* II) modules were joined to the VH (*Eco* RV and *Apa* I) and Vκ (*Bam* HI and *Hind* III) modules, respectively, in plasmid vector pVITRO1*neo*. Each cDNA contained a Kozak box. Plasmids of chimeric P1G4 hIgG1, hIgG2,

isolated from a patient diagnosed with a tumor of 5.5 cc in size [20].

small cell carcinoma and 2.9% of mixed carcinoma type [42].

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

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

#### **Figure 4.**

*Advances in Precision Medicine Oncology*

**5. AGR2 in bladder cancer and lung cancer**

to AGR2+

Although 25% primary bladder tumors of a study cohort were found positive for AGR2, this percentage increased to 45% in the synchronous lymph node metastases [18]. The discordance between primary cancer and lymph node metastasis could involve a phenotypic change [39], which could occur in bladder cancer cells going

*AGR2 in prostate cancer metastases. Tumor cells in distal metastases – bone (top left), liver (top right), lymph node (bottom left) – are strongly stained for AGR2. A metastasis containing small cell carcinoma (bottom* 

link to patient survival in one cohort analyzed [18]. A role for CD10 in lymph node spread of bladder cancer was also not apparent [18]. Recently, UW Urology initiated the bladder cancer rapid autopsy program modeled on the Department's success in obtaining>40 prostate cancer LuCaP lines with various characteristics [40], and has established lines, dubbed CoCaB, from primary and metastatic urothelial cancer. To date, seven lines are available: CoCaB 1, CoCaB 8 urothelial carcinoma, CoCaB 11, CoCaB 19 squamous carcinoma, CoCaB 12 urothelial with sarcomatoid component; three lines from metastases of deceased patients: CoCaB 10 (liver metastasis) urothelial carcinoma, CoCaB 14.1 (omentum metastasis) and CoCaB 14.2 (liver metastasis) urothelial carcinoma with squamous features. These lines were profiled by RNAseq and exome sequencing. Notably, the lines derived from metastases showed high AGR2 expression (**Figure 4**). This is in line with our result on prostate cancer metastases where, except for small cell carcinoma, all tumors as well as the LuCaP lines derived from metastases showed high AGR2 expression [27]. Likewise,

pancreatic cancer metastases invariably showed high AGR2 expression [37]. In non-small cell lung cancer, higher AGR2 expression in primary tumors is associated with a poorer outcome for patients under 65 [14, 19], though over 90%

cancer. Thus, depending on the microenvironment, the metastatic function of AGR2 is not predominant in differentiated prostate tumors. When cancer cells have escaped the prostatic capsule (via CD10), AGR2 appears to be essential for distal

. AGR2 in bladder cancer, unlike prostate cancer, showed no

(**Figure 2**). This is in contrast to the finding for prostate

**100**

of the tumors are AGR2+

from AGR2−

**Figure 3.**

*right) is not stained.*

*Bladder cancer cell types. Cancer cell lines are identified on the x-axis (CoCaB and uc). Histogram represents fold in expression difference as scored by RNAseq signal values on the y-axis for MME/CD10, AGR2, XIST (X-inactive specific transcript) and CD24. The two CoCaB lines established from metastases are indicated by small red oval.*

colonization of other organs for prostate cancer cells. In the disease course, AGR2 expression could be down- or up-regulated in cancer cells through mechanisms yet to be elucidated.

A meta-analysis of the published data on the clinical utility of AGR2 expression in various solid tumors summarizes the link to survival findings [41]. AGR2 expression in primary cancer can be associated with better survival for prostate cancer, no survival advantage in bladder cancer, poorer survival for lung cancer. While AGR2 in pancreatic cancer could have a significant impact in treating both early and advanced diseases, anti-AGR2 therapy in prostate cancer could prove promising in treating advanced disease as 96.4% metastatic lesions of adenocarcinoma being AGR2+ against 0.7% of AGR2− small cell carcinoma and 2.9% of mixed carcinoma type [42].

### **6. Chimeric human:mouse AGR2 antibodies**

Mouse monoclonal antibodies to AGR2 were generated by inoculating bacterially produced recombinant AGR2 in RBF/DnJ mice [20]. Two of eleven clones collected, P1G4 (mIgG1) and P3A5 (mIgG2a), were tested positive for binding to native AGR2. These antibodies perform well in immunostaining of frozen tissue sections but not formalin-fixed tissue. ELISA based on these two antibodies was able to detect 17 pg of cancer-secreted AGR2 in 100 μg of total urinary protein isolated from a patient diagnosed with a tumor of 5.5 cc in size [20].

Based on our published design [43], the mouse antibodies were converted to chimeric human:mouse. The mouse variable VH and Vκ of P3A5 and P1G4 sequences were determined via reverse transcriptase-polymerase chain reaction (RT-PCR) with designed primers from mIg mRNA isolated from the respective hybridoma cell lines. The V sequences were-matched to known murine Ighv and Igkv genes. The human constant Cγ and Cκ domain cDNA were cloned by using designed primers from white blood cells of healthy donors. The hIg cDNA were verified by DNA sequencing and restriction enzyme digestion. The Cγ (digested by *Apa* I and *Bam* HI) and Cκ (*Hind* III and *Avr* II) modules were joined to the VH (*Eco* RV and *Apa* I) and Vκ (*Bam* HI and *Hind* III) modules, respectively, in plasmid vector pVITRO1*neo*. Each cDNA contained a Kozak box. Plasmids of chimeric P1G4 hIgG1, hIgG2,

hIgG4; P3A5 hIgG1, hIgG2, hIgG4 were generated [22]. The different Cγ plasmids could be distinguished by *Sac* II and *Eco* RI digestion. Cγ3 sequences were not detected in the cDNA prepared from 1 ml of blood.

Human embryonic kidney fibroblasts, HEK293F, were transfected by the hIgG plasmids, selected for G418 (neomycin) resistance and cloned. RT-PCR analysis of the transfected cells showed equivalent mRNA levels for the 560 bp neo, 720 bp IgL and 1420 bp IgH [22]. The culture media supernatant was assayed for AGR2 binding. P1G4 was used to capture AGR2 (secreted by eAGR2<sup>+</sup> prostate cancer LuCaP 147 in tissue collagenase digestion media to obtain single cells from minced tumor pieces) followed by P3A5 (positive control) and culture supernatant containing the chimeric antibodies. HRP-conjugated anti-mIgG2a or anti-hIgG were used for detection. The chimeric antibodies were found similar to P3A5 in AGR2 binding [22]. Untransfected 293F cells or transfected with a defective L-chain construct produced no binding. Media from serial culture passages showed that hIgG synthesis continued indicating stable integration of the transgenes into the host genome. The hIgG-producing clones were weaned from fetal bovine serum supplement, and cultured in the absence of drug for G418 is toxic. The serum-free growth media contained few other proteins (293F cells are non-secretory fibroblasts compared to hybridoma cells), and a spin-filtration step using a 30 K molecular weight cut-off could readily concentrate the 150 kDa IgG proteins [22]. If necessary, each antibody can be purified further on protein G-sepharose. The chimeric hIgG1, hIgG2, hIgG4 and mIgG2a P3A5 detected similar amounts of AGR2 secreted by different LuCaP lines: 147, 35CR, 86.2, 105 (**Figure 5**). Thus, these chimeric antibodies retain specific antigen binding and can be produced more economically via large-scale cell culture (instead that of hybridoma cells, which also produce a defective non-specific mouse light chain, and potentially infectious murine bioactive agents).

To increase the production of hIgG by transfected 293F cells, we transferred the different hIgG gene cassettes into plasmid vector pVITRO1*bsr* (encoding blasticidin resistance). Selected hIgG-producing neo<sup>R</sup> clones were transfected by the *bsr* constructs and selected for blasticidin resistance. The resultant neoR bsrR clones showed increased amounts of secreted hIgG in the culture media due to the increased gene dosage [22].

#### **Figure 5.**

*Chimeric IgG binding to AGR2. Both chimeric (transfected HEK clone p13–1) and P3A5 detect varying amounts of AGR2 secreted by different LuCaP PDX lines. Clone p12–1 is a defective construct that produced untranslated L chain mRNA. Similar binding was also shown by the other IgG types. The absorbance units of ELISA are on the y-axis.*

**103**

**Figure 6.**

*tumors (\*) in two C57BL/6 mice.*

eAGR2+

Ki67 indicated less (AGR2+

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

our obtained monoclonal antibodies. The mouse Agr2+

**7. Cancer cell surface expression of eAGR2 and tumor localization**

[44], derived from an engineered C57BL/6 mouse strain to develop pancreatic cancer [45], was incubated with P3A5 followed by dye-conjugated anti-mIgG2a. Antibody binding to the cell surface was indicated by fluorescence shift (*vs*. isotypematched control) [22]. Most available anti-human AGR2 antibodies, like P3A5, recognize both human AGR2 and murine Agr2 as the two proteins share a high degree of sequence homology. Human pancreatic cells were previously shown to

have cell surface expression of eAGR2 using a different antibody [37].

be obtained in human patients, i.e., specific localization to eAGR2<sup>+</sup>

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

To demonstrate cancer cell surface expression, we used flow cytometry with

To demonstrate tumor localization, 111In-radiolabeled P3A5 was injected into mice carrying DT6606 tumors. At 48 h post-injection, the implanted tumors showed intense labeling (**Figure 6**). There was minimal labeling of the iAgr2<sup>+</sup> bladder or lung, or elsewhere [22]. The imaging confirmed cancer cell surface localization as well as cancer cell specificity of eAgr2. The cross-reactivity between human AGR2 and mouse Agr2 allowed one to speculate that a similar result would

there is a strong likelihood that ant-AGR2 would be highly effective against cancer

Our P1G4 antibody was found to possess a clinically useful property. Using

 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

fested 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

*Tumor localization of P3A5. The SPECT/CT scans show specific uptake of radiolabeled P3A5 by Agr2<sup>+</sup>*

pancreatic cell line DT6606

pancreatic (or

 *DT606* 

organs. With cancer-specific localization,

) tumor cell proliferation in P1 + Gem, which was mani-

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

other solid) tumors and not to iAGR2<sup>+</sup>

with minimal reactivity towards non-cancer tissue.

*Advances in Precision Medicine Oncology*

detected in the cDNA prepared from 1 ml of blood.

binding. P1G4 was used to capture AGR2 (secreted by eAGR2<sup>+</sup>

light chain, and potentially infectious murine bioactive agents).

structs and selected for blasticidin resistance. The resultant neoR

resistance). Selected hIgG-producing neo<sup>R</sup>

dosage [22].

To increase the production of hIgG by transfected 293F cells, we transferred the different hIgG gene cassettes into plasmid vector pVITRO1*bsr* (encoding blasticidin

increased amounts of secreted hIgG in the culture media due to the increased gene

*Chimeric IgG binding to AGR2. Both chimeric (transfected HEK clone p13–1) and P3A5 detect varying amounts of AGR2 secreted by different LuCaP PDX lines. Clone p12–1 is a defective construct that produced untranslated L chain mRNA. Similar binding was also shown by the other IgG types. The absorbance units of* 

clones were transfected by the *bsr* con-

bsrR

clones showed

hIgG4; P3A5 hIgG1, hIgG2, hIgG4 were generated [22]. The different Cγ plasmids could be distinguished by *Sac* II and *Eco* RI digestion. Cγ3 sequences were not

Human embryonic kidney fibroblasts, HEK293F, were transfected by the hIgG plasmids, selected for G418 (neomycin) resistance and cloned. RT-PCR analysis of the transfected cells showed equivalent mRNA levels for the 560 bp neo, 720 bp IgL and 1420 bp IgH [22]. The culture media supernatant was assayed for AGR2

LuCaP 147 in tissue collagenase digestion media to obtain single cells from minced tumor pieces) followed by P3A5 (positive control) and culture supernatant containing the chimeric antibodies. HRP-conjugated anti-mIgG2a or anti-hIgG were used for detection. The chimeric antibodies were found similar to P3A5 in AGR2 binding [22]. Untransfected 293F cells or transfected with a defective L-chain construct produced no binding. Media from serial culture passages showed that hIgG synthesis continued indicating stable integration of the transgenes into the host genome. The hIgG-producing clones were weaned from fetal bovine serum supplement, and cultured in the absence of drug for G418 is toxic. The serum-free growth media contained few other proteins (293F cells are non-secretory fibroblasts compared to hybridoma cells), and a spin-filtration step using a 30 K molecular weight cut-off could readily concentrate the 150 kDa IgG proteins [22]. If necessary, each antibody can be purified further on protein G-sepharose. The chimeric hIgG1, hIgG2, hIgG4 and mIgG2a P3A5 detected similar amounts of AGR2 secreted by different LuCaP lines: 147, 35CR, 86.2, 105 (**Figure 5**). Thus, these chimeric antibodies retain specific antigen binding and can be produced more economically via large-scale cell culture (instead that of hybridoma cells, which also produce a defective non-specific mouse

prostate cancer

**102**

**Figure 5.**

*ELISA are on the y-axis.*
